Pharynaceutca Research, Vol. 26, No. 10, October 2009 (© 2008) DOr LUIOoTSOSoORTS Research Paper Permeation Enhancer-Containing Water-In-Oil Nanoemulsions as Carriers for Intrayesical Cisplatin Delivery ‘Tsong-Long Hwang,' Chia-Lang Fang,” Chao-Huang Chen, and Jia-You Fang** Received May 12,2009; cceped July 2, 2009; published oniine Avggst 4, 2009 Purpose, In the present work, we developed waterivoll (wo) nanoemulsions for administration of cxplatin. ‘Methods. The nanccmuisions were made up of soybean oil as the oil phase and Span ly Tween 80, or [Br 98 asthe emulsifer system. eeTerpincol and oleic ack were incorporated as permcation enbancers. The piysicctemical duracterisics of droplet siz, zet potential, and viscosity were determined. \Nanoemulsions were administered intravescally for 14 h to rats iv vivo, Animals were subsequently ‘sctifoed, and the bladers were harsted fo examine drug accumulation and histology. Results Ranges ofthe mean size and eta potential were 30~90 nm and ~34 to ~9.3 mV, respectively. ‘The akon of enhancers futher reduced the siz of dhe nanoemlsions, The vscisity Of all ystems exhibited Newionian behavior. The dspltinehadod renocmulsions were active against bladder cancer elk. The nancemubions with Brj 98 exhibited the complet inhibition of cell probferation. The encapsulation of cisplatin and carboplatin, another derivative of cisplatin, in nanoemmlsons resulted in slower and moresustained release. The amount of drug which permeated into bladder tues Significantly inereased when using caters containing Brij 98 withthe ceterpincokcontaining, formulation showing the best result The nanoemukion with avterpineol prolonged the duration of higher drug accumulation to 3~4 by At the later stage of administration (3~4 bh), this system increased the bladder ‘wal deposition of ciplatin and carboplatin by 24~33}fold compared to the canto solution, Histological examination of the rothelium showed nearnormal morphology in ras insiled with dese nano intravesical mubio Tena posi cause sight dsumaton of uni cs ‘nanoemulsions are feasible t loa cisplatin for intravesical dru Conckisions: The very. [KEY WORDS: bladder, cisplatin; inmnovesical delivery; nancemulion; sstaied reese. INTRODUCTION An estimated 261,000 new cases of bladder cancer are diagnosed worklwide each year (1), Bladder cancer is the fourth mest common malignancy among men, but the high recurrence rates likely make it the most prevalent malignancy among all cancers, and certainly the most expensive per patient treated (2.3). More than 70% of bladder cancers [present as nonemusclosinvasive bladder cancer (NMIBC) (4). ‘The current treatment consists of transurethral resection (TUR) of visible tumors, followed by intravesical chemo- therapy to reduce disease reourence and progression (5), ‘The rationale for intravesical therapy is to maximize the exposure of tumors located in the bladder cavity t0 ther= "Cel Phamacolbey Laboratory, Graduate Instinute of Natural Prociuctss Chang, Gung Universi, Kowishan, Taoyuan, Taivan, *Depurtment of Pathology, Colle of Mecicine, Taipei Medical University Taipei, Taran, 2 Pharmaceutics Laboratory, Graduate Imtitute of Natural Proc, Gang Gung, Universi 259 Wen-Hiwa Ist Road, Kweishan, Taoyuen 333 Taiven. “To whom correspondence should be adresed. (eal fajy@nil caueduutw) (MeesCALNDICAIAO 1 AD Sir Sen + Bess Mea LC peutic agents while limiting the systemic exposure and. thereby limiting toxicity to the host. However, intravesical delivery hns to overcome its own set of challenges, and most prominent among them is the low residence time of a drug in the bladder, which necessitates frequent instillation (6). ‘Nanosystems with a well-defined particle size and shape have immense potential for intravesical delivery, as they can enhance the ability of drugs to cross the urothelium, A higher surfaco-to-volume ratio may also be responsible fer increased, absomption of encapsulated drues (7). Goplatin is widely used for treating bladder cancer (S). Given the tonicity and mortality associuted with cispkatin-based. chemotherapy, intravesical administation would signifcantly reduce the systemic side effects, thus improving its therapeutic, index (S10), Csplatin is a hyelrephilic molecule with an n= ‘octanol water partitioning coefficient (ogP,ssunaystes) F219. ‘The efficacy of intravesical therapy for bladder cancer isin part, limited by the poor permeation of hydrophilic drugs into the ‘urothelium. Developing a formulation for intravesical cisplatin delivery not only for higher concentrations in the blader wall, but alo for longer retention with reduced sie effects woul be very beneficial, Water-ineil (Wwio) nancemulsions, a class of naniosystems with droplet ses of 20200 nm, are considered. to be enhanced, proonged-elease systems for hydrophilic BidPermeation Enbancer-Containing Watern-Oil Nanoemulsions as Carriers for Intravesical Cplatin Delivery drugs with the oil and interfacial layers acting as release bartiers (11412), Nanogystems may be suitable for cisplatin 10 achieve these aims, “The purpose of this work was to develop wio nano ‘emulsions as intravesical delivery systems to efficiently target, cisplatin to the bladder. Permeation enhancers, such as c= terpineol and oleic acid, were abso loaded in the nano emulsions, since the chemical enhancers can increase the permeability of drugs through the urothelium (13). Thus, the anti-cancer activity of cisplatin can be enhanced via the encapsulation into the nancemusions. The droplet siz, zeta potential, and viscosity were determined as the physicochem= ical characteristics of the nanoemukions. The cytotoxicity of the cisplatin-loaded carriers against urothelial carcinoma cells, was also determined, The drug release was evaluated using an in vioo Franz diffusion assembly. Drug permeation into the bladder wall was examined using the rat as an animal model. Finally, possible mechanisms of the efficiency of intravesical cisplatin delivery by nanoemulsions were explored. ‘MATERIALS AND METHODS ‘Materials Ceplatin, carboplatin, uoreswvin isthincyanate (FITC), saybean oil, Span 82, octerpineol, and oleic acid were pure chased! from Sizma-Akitich Chemical (St. Louis, MO, USA). “Tween 80 was obtained from Kanto Chemical CTokyo, Japan). Bij 98 was provided by Actos Organs (Geel, Belgium). Preparation of Nanoemubsions, The oil and aqueous phases were separately prepared. ‘The oil phase consisted of soybean oil and emulsifiers, while the aqueous phase consisted of water and the dnig (10 achieve a concentration of 13 mM in the final proxiust). The two phases were heated separately to 55°C. The oil phase was further mixed using a high-shear homogenizer (Pro250, Pro Scientific, Monroe, CT, USA) for 10 min, Then the oll phase was acded to the aqueous phase and sonicated using a probe-type sonicator (VCX600, Sonics and Materials, Newtown, CT, USA) for 15 min at 35 W. Compositions of the nancemulsions are listed in Table [. ‘Determination of the Size and Zeta Potential ‘The mean vesicle size (zaaverage) and zeta potential of the nanoemulsions were measured by photon correlation spectroscopy (Malvern Nano ZS® 90, Worcestershire, UK) using a heliumneon laser with a wavelength of 633 nm, The fommuktions were diluted Sold with soybean oil before 2315 the measurement, Photon correlations of spectroscopic ‘measurements Were carried out at a scattering angle of 90°, Detennination of Viscosity ‘The visoesity of the nanoemulsions was measured as the shear stress (Pa) as a function of the shear rate (I) using a Carti-Med CSL? 100 rheometer (TA. Instruments, New Castle, DE, USA). The diameters of both the cone and plate spindle were 60 mm. The determination mode was set to flowsstep measurements with shear rates of 0~ 1000's. The cone angle used for the measurements was 2°, Cytotoxicity Asay ‘The T24 human urothelial carcinoma cell line was purchased from American Type Culture Collection (Rock- Ville, MD, USA), Cells were seeded at an inital concen tration of 2x 10" cellsivell in a 244Well plate and incubated in 1 mi of medium (10% fetal bovine serum, 9% RPMI 1640, and 1% penicllinestreptomycin). Water and the nanoemulsions with or without cisplatin diluted with ‘medium were added at 24 post-inoculation, and plates were incubated in a 5% COy atmosphere at 37°C. Subsequently, the medium was incubated for 72 h. After ‘washing with phosphate-buffered saline (PBS), cells were incubated with 5 mg/ml 3[4S-limethylthiazol-2-y1]-25- diphenybtetrazolium bromide (MITT) in RPMI 1640 for 2h at 37°C, Formavan anstals resulting from the reduction of MIT were dissolved by ackling 200 jul DMSO and gently agitated for 30 min, The absorbance of the supernatant was then measured jometrically in an enzyme-linked immunosorbent assay (ELISA) reader at 550 nm. Cell Viability was cakulated asa percentage of the control. Partition Coefficient (ogPeayrac) of the Drug between the Oil and Water Phases The partition coefficient was determined by equiibrating chug partitioning between the oil and water phases Cisplatin or carboplatin at QS mg was dispersed in a mixture of soybean oil (1 ml) and water (I ml) and shaken for 30 min at 37°C. The aqueous phase was subsequently filtered through a polyvinylidene diftuoride (PVDF) membrane with a pore size (of (45 jum. The drug.concentration in the aqueous phase was determined by atomic absorption spectrophotometry (AAS: Z-S10), Hitachi, Tokyo, Japan). In Vitro Drug Release from the Nanoemulsions Ggplatin or carboplatin release from the wo nano- emulsions was measured using a Franz diffusion cell, A. ‘Table 1, The compositions ant their percents (i, wiv) of waterieil mnoemulsions used in this study Coke Water Soybean cil ‘Span 80 Tween 8 Bri 98 o-Terpineol Oleic add Ni 5 0 » 2 = = = nN 8 5% » = B = = NB 8 45 » = 2 5 Nt 8 45 » = 2 22316 cellulose membrane (CelluSep® 73, with a molecular weight cutoff of 2500) was mounted between the donor and receptor compartments, The donor medium consisted of 0S ml of rehicle containing drugs. The receptor medium (55 ml) consisted of pH 74 citrate-phosphate bufier. The available diffusion area between the cells was 0.785 en. The stirring rate and temperature were set to 600 rpm and 37°C, respectively, At appropriate intervals, 300441 aliquots of the receptor medium were withdrawn and immediately replaced ‘with an equal volume of fresh buffer. The amount of drug released was determined by AAS. In the study examining the influence of urine on drug reas, (5 ml of synthetic urine was added to the donor compartment. The synthetic urine solution contained 14,1 g NaCl, 28 g KCL 173 g urea, 1.9 ml of a 25% (vA) ammonia solution, 046 g CaCl and O43 g of ‘MeSO4 made up to 1 L with water (14). In Vivo Intravesical Delivery Female Sprague-Dawley rats weighing 225~250 g were used in ths study. The animal protocol was approved by the Institutional Animal Care and Use Committee of Chang Gung University (Taoyuan, Taiwan) Animals were subcuta- neously anesthetized with urethane (12 g/kg) before intrae \esical instillation. Residual urine was evacuated by pressing the lower abdomen. A polyurethane catheter (BD Angiocath Plus®, Becton Dickinson Korea, Gyeonebuk, Korea) was inserted into the bladder. The bladder was washed with 0 ml of normal saline. Subsequently, 0.2 ml of nanoemulsions containing cisplatin or carboplatin was instilled into the blader. The catheter was removed, and the urethra was ligated with cotton thread, The dwell times for instillation were 1,2, 3, and 4 h, After the application time, the bladder was excised and washed with sine, The drug in the bladder tissue was extracted by a homogenization method. TWO milliliters of 431% sulfosali- oylic acid was added to the excised tissue in a glass homogenizer and ground for 5 min. The resulting sokution ‘was centrifuged for 15 min at 12000 mpm. The drug content in the supernatant was measured by AS, ‘Confocal Laser Scanning Microscopy (CLSM) Localization of FITC within the bladder wall. was determined by CLSM after vivo intravesical delivery, FITC inthe vehicles ata concentration of 10 iM was used. After in Vivo instillation for 3 by the bladder samples were excised to examine the fluoresoem images. The thickness ofthe bladder ‘wall was optically scanned at ~ 104m increments through the Zan of a Leica TCS SP2 confocal microscope (Wetzlar, Gemnany). Optical excitation was carried out with a 488m angon laser, and the fluorescence emission was detected at 496-559 mm, Histological Beamination of the Bladder Immediately fer treatment with an aqueous solution or the mnoemulsions by intravesical instillation for 3h, a specimen of the exposed area was taken for histological examination, Each specimen was fowed in a 10% buffered formaldehyde solution (pH 7.4) for at least 48 h and stained ‘Hwang, Fang, Chen and Fang with hematoxylin and eosin, For each simple, three different sites were examined and evaluated under light microscopy (Eclipse 4000, Nikon, Tokyo, Japan). ‘Statist ‘ Statistical analysis of differences between different treat= ‘ments was performed using unpaired Student's test. A 0.05 level of probability was taken as the Kyl of significance. An. analysis of variance (ANOVA) was also used if necessary. RESULTS Physicochemical Characterization of the Nanoemubions The drug delivery potential of the nanoemulsions may depend upon the droplet characteristics, including thei size, charge, hydrophilicity, and viscosity. The present formulation (N1) was stabilized by a combination of two emulsifiers: a sugar ester of sorbitan monookeate (Span 80) andl a polyoxy= ethylene 20 sorbitan monooleate (Tween 80). The ingredients and their percentages of this formulation were designed and ‘modified from the previous study by Wu er a, (I1) for skin delivery purpose. A pseulo-ternary phase diagram with a stable percentage range of oil, water, and surfactant mixtures is determined by the nanoemulsions developed in this work (1), The substitution of Tween 80 (with a hydrophile- lipophile balance (FHLB) of 15) with another polyoxyethylene surfactant, Brij98 (with an HLB of 152), also formed a stable emulsion system (ND). The size analysis indicated that the nanoemulsions with Span 8 and Tween 8) (NI) had mean droplet sizes of 90.0 nm, while that of the N2 nanoemulsion was 55.6 nm (Table D, The absolute zeta potential of the ‘Tween S-containing formulation (NI) was~93 mV, Replace ‘mentof the hydrophilicnon-ionic emubsifier of Tween $0 with Brij 98 (N2) led to an initial decrease in the 2eta potential (p< (0.05). The Brij 98-containing nanoemulsion (N2) was chosen for further formulation design to which the enhancers were added, The addition of acterpineol and oleic acl proxiuced nanoemulsions (N3 and N4) with reduced sizes of 277 and 454 nm, respectively (p<0.05). On the other hand, the zeta potential did not change after a-terpineol incorporation (N2 1s. N3, p>0.05). The adltion of oleic acid (N4) contributed to a slight but significant increase (p<0.05) in the negative zeta potential. A preliminary stability test had demonstrated fan insignificant change (p=005) of mean size anki zeta potential within 30 days of storize at 37°C and 65% relative humidity Gata not shown). ‘The apparent viscosity of the nanoemubions was calcu lated from values of the shear stess and shear rate on the ‘upward curve shown in Fig. 1. The appearance of the curves confirms the Newtonian behavior of the investigated formu lations, Tween S-containing nanoemulsion (NI) showed the highest viscosity, followed by N4, N2,and N3, Cytoroxicity Asay ‘The present work evaluated the efficacy of the nano emulsions of delivering cisplatin to urothelium cancer cells by examining their cytotoxicity. As shown in Fig, 2, the viability, Of the urothelium cancer cell line treated with fee cisplatinPermeation Enhancer-Containing Water4n-Oil Nanoemulsions as Carriers for Infravesical Cisplatin Delivery 1. so} |— ts Ho 217 at a shear rao (Vs) Fig. L. The vicosity of nanoemusions measured by shear tess (Pa) with increasing shear rates of O~ 1010S (2 uM) was ~32%. Cisplatin loaded in the mnoemulsions showed higher cytotoxic activity (p<(05) than the free drug ina 724 incubation experiment, Nanoemulsions with Brij 98 (ND-N) completely inhibited the growth of urothelial carcinoma cells The incorporation of enhancers into the ranoemulsions did not influence cisplatin’ activity against cancer cells. Empty nanoemulsions without cisplatin. were also tested, and it was found that the systems themselves had no effect on the cytotoxicity. This indicates thatthe cytotos- icity toward the urothelium carcinoma cells was mainly a consequence of the cisplatin molecules, In Vitro Gsplatin Release from the Nanoemulsions, In order 1© develop a prolonged release stem with general applicability tf of great importance to understand, the release mechanisms and kinetics. The in vit release behaviors of cisplatin from the aqueous solution and nano- emulsions are shown in Fig. 3A. Release of cisplatin from the solution tee form) was rapid, With 97% released after 8 hat 37°C. The rekae kinctics of cpatin from the ranoemule sions exhibited slower, more-continuous release for 8h The calvetay ©) 8 cam oe Fig, 2 Viability (%) of human 124 urothelial carcinoma cells following treatment with free cisplatin in water and cisplatindaded smancemulsions. Fach value represents the mean = SD of ive experiments. Teh) ig. 3. Jn vitro release percentage (6) of cisplatin acres celtlese ‘membrane from doubke0.05) in the presence of urine, In Vivo Intravesical Cisplatin Delivery Rats received an intravesical dose of cispkatin to examine the amounts in the blacker wall during 4h of administration. Fig. 4 shows the time profile of cisplatin concentration in the tissue. A steeper decrease of the cisplatin concentration was observed in tissue segments following an increase in the application time. ‘The results indicate that higher cisplatin1 2 3 7% Tinet) Fig. 44 Jn vivo ceplatin accumulation (nmol/g) in bladder tsues ater intavesical instillation of water (free ool) and nanoemubions ‘with dspltin for 1~4 h, All data are presenta asthe mean = SD of sx experiments * Significantly higher (<0.05) compared to the ccontral at the same time point; * significantly lower (p<005) ‘compared tothe contol at the same time point. concentrations were obtained in the bladder wall when admin- ‘stering nanoemulsions with Bri 98 (N2 to N4) compared to the free control solution, On the other hand, systems with Tween 80 (N1) showed the lowest drug accumulation (p<005) in tisues ‘among all formulations examined. a-Terpineo! incorporation increased the drug penetration (p0.05) at 3 h, whereas oleic ack haxl no measurable effect (p>005) at this time point. All formulations exhibited comparable cisplatin accumulations at the ast time point 4). (Carboplatin as a Model Drug for Nanoemulsions Specific groups of patients, such as the elderly and ‘patients with renal dysfunction or a poor performance status for co-morbidities who cannot tolerate cisplatin therapy, shoukl receive carbopiatin-based treatment (8). Carboplatin is selected mainly because of its lower non-hematological toxicity compared to cisplatin. The delivery of carboplatin from the nanoemulsions was aso tested. As shown in Fig, 5, release of carboplatin from the nanoemulsions was sustained with tren similar to those of cisplatin. The action of urine to the carers produced an approximately equivalent release of carboplatin for all nanoemulsions (Fig. SB). In the in vivo cirug permeation study, the free carboplatin solution showed higher accumulation (p<0.05) in the tissue compared to free splatin as observed in Fig, 6, N3 was selected as the nanoemuion to examine carboplatin absorption because of its good! performance of c&pltin deposition. The tissue level of carboplatin after nanoemulsion application was signif cantly higher (p<0.05) than that with free carboplatin application at 2~4 h, The system yiekled a bladder tissue concentration of 12~16 nmol/g during 4 h of administration, Steadyestate carboplatin accumulation was thus achieved. (Confocal Laser Scanning Microscopy (CLSM) CLSM was used to observe the superficial FITC istribution in the blackler wall. Because of the high hyeto- ‘Hwang, Fang, Chen and Fang philicty of FITC, it likely resided in the inner phases The superficial layer of nine fragments was optically scanned at ~ 10-4 increments from the surface of the mucesa as shown. in Fig, 7. The total ~70 ym comprises the urothelium and a part of the lamina propria. Fig, 7A illustrates the results obiained from CLSM following intravesical delivery of free FITC in an aqucous solution for 3h. There was no or negligible green fluorescence in the images of the free control, It can be seen that the intensity’ of the green fluorescence was much greater with the N2 formulation (Fig. 78). The fluorescence in the horizontal section at 27~ 46 um was strong, for the N2 system, then the intensity sgraduelly decreased with increasing depth. a-Terpineol in the nanoemulsion (NG) further enhanced the signals to a more intense level (Fig. 7), FITC loaded in the N3 formulation pemeated the deeper layers. The signals were even stronssr in deeper regions of the tissue. Histological Examination of the Bladder Light microscopy was applied 10 evaluate the influence of the nanoemubsions on the morphological properties of the bladder wall. As shown in Fig. SA, the bladder tissue remained intact after exposure to water for 3 h (control) A) ame f) Fig. 8 Jn vitro rekase percentage (%) of ebopkain acess a calulose membrane from doublolstiled water (control) and nano ‘emulsions (A) and in the presence of symhetic urine (0.5 ml) in the donor compartment (B). All datrare presented as the mean = SD of, {our experiments.Permeation Enbancer-Containing Watern-Oil Nanoemulsions as Carriers for Intravesical Cplatin Delivery 8 » 0 Amourtin Hacer sae (ree) Time) Fig. @ Jn vivo cutoplatin accumulation (nmol) ia bladder tissues ie intnesial instillation of water (free control) and aetexpineok ‘containing nanoemubion (NS) with carboplatin for ~4 fy AI data are presented as the mean = SD of six experiments * Significantly higher (p<005) compared to the control atthe sime time pains * significant lower (p<005) compared to the contro atthe Sime time point. 2319 ‘The results of microscopy revealed that the morphology of tissue exposed to N2 showed no changes (Fig, SB). Intravesical insillation of the nanoemulsion with e-terpineol (N3) revealed histological changes as shown in Fig. SC. Some detached ‘aperfical urothelium was noted (arrows), which may have resulted from the desquamation of umbrella cells. Some congestion wasalso observed inthe blood vessels. These changes, ‘were due to early, miki disruption of the bladder structure. DISCUSSION Despite intravesical chemotherapy being used after TUR, Of superficial blackler tumors, the recurrence rate is stil 6% ~44% (15). The incomplete response often seen with the Use of conventional formulations for intravesical therapy can be partly attributed to resistant drug targets or unsuccessful rug delivery to diseased bladder tissue (7), In the current ‘Study, wio nanoemulsions with an optimal formulation design provided enhanced and controlled cisplatin and carboplatin delivery to the bladder. Sustained release of the drug was observed afer nancemukion encapsulation. Droplet sizes of the present formulations ranged 30~ 90 nm, which fall in the range of so-called nanoemulsions ig. 7. Confocal laser scanning micescopic (CLSM) micograpls of rat bladder tissues after dhe av vio intravesical insilltion of FITC for 3h from (A) water (fre control), (8) cemulsion (N3) (oi thoueh the Z-axis from the sur wanoemukion with Brij 9% (NI), and (C) the aserpineole mupnifiction, 209). The specimen was viewed by CLSM at ~l04um increments eto the decper kyers (op to botlam).1A) ‘Hang, Fang, Chen and Fang Fig, 8 Histobgical examination of rat blacker tisues after ir vivo intravesical insilation for 3h with CA) water (contro), (B) menoemulsion with Bui 98 CN), and (C) c-terpincol-confaning nanoemubion (N3) (original magnification, 100%). (20-200 nim). All formulations were <100 nm, which is advantageous since the emulsions can be sterilized simply by passing them through a sterile syingeriven filter with no need for thermal treatment (16,17). The incorporation of Brij 98 (N2) instead of Tween 8) (N1) led to a significant reduction in the droplet siz. Brij 98 may have the ability to strengthen droplet integrity (Is), resulting in strong cohesion of the droplets and the avoidance of aggregation. All emukifiers used here were nonionic species. The negative charge in the interface is believed 10 be the result of the ionization of the free fatty acids derived from the hydrolysis of triglycerides in soybean oil The larger surface area in the larger inner phases may provide a higher surface potential (13). This can exphin the higher zeta potential of the Tween ‘SO-containing nanoemulsion (NL) compared to the Brij 9S containing one (N2), The first barrier to drug diffusion in blacker tisue i the highly negatively changed glycosamino- glycan layer and membrane plaque, which prevent drugs from reaching the urothelium (19). The NI system may have been unfavorable for interactions with the blader wall because of its higher negative charge. On the other hand, interfacial tension tends to be lower for emulsions with smaller droplet sizes (20). rendering their permeation easier. Thereby, the N2 system was selected for further formulation design, ‘The audition of enhancers reduced the mean size of the nanoemulsions, especially that with ceterpineol (N3). The logPosscuwacr Values of o-terpineol and oleic acid are 3.1~ 33 and 7, respectively (21). The discrepancy between the polarity ofthe enhancer-containing oil phase and the aqueous phase was smaller for acterpineol. Stabilization and mixing of the two phases with these emulifiers were easier with the ccterpineol-containing system resulting in the production of smaller droplet sizes, Alkali fatty acids, such as oleic ace, are thought to yield mixed films with a higher packing density of interfacial film-forming components (22). Adding okeicacil 10 the systems may increase the negative zeta potential as observed in the present study. Local retention conditions in tissues are an important parameter for drug delivery by emulsions (23). The viscosity! theology properties are factors affecting the retention ability Larger crops have higher viscosities (20). This phenom- {enon is identical to the present results in which the Tween 80- containing formulation (NI) showed a greater viscosity than the Brij 9S-containing one (N2). Another possible reason is the potency of the water swelling of Tween 80 to increase the apparent viscosity (24), The cytotoxicity results showed that free cisplatin induced proliferation inhibition in urothelium cancer cells Cisplatin was slowly released from the nanoemulsion drop= Jets. Henee the high activity against cultured cells was not totally due to the direct penetration of free drug into the cytoplasm, One possibility is the advantage of the oily extemal phase of the nanoemulsions increasing drug absomp- tion into tumor cells 25). Another possiblity is that cellular uptake of the crug can occur by an endocytotic pathway’ of droplets or by fusion of the surfaces with the cell membrane, leading to increased internalization of the droplets and drug release inside the tumor (2627). A detailed investigation ofPermeation Enbancer-Containing Watern-Oil Nanoemulsions as Carriers for Intravesical Cplatin Delivery the nanogstem’s effect on oytotaxicity wasnot the main focus of this study. Further elucidation of the meckanism will be attempted in future studies. Assuming that cisplatin released from the nanosystems still play an essential role in cytotox icity (78), the lover cytotoxicity of cisplatin in mnoemulsions with Tween 80 (NI) than the other carriers may be consistent with the relatively slower release profile of the drug. Gisplatin is a platinum-chelated complex with four ligands two ammonias and two chlorides, The wo chloride ligands are gradually substituted with HO. molecules in water. Both Pt(NHs):Cl(OH3)* and. Pt(NHs)2COHD):"* can be formed by the aqueous complex (29,20). The partition coefficient of cisplatin between soybean oil and Water was determined, The logP,asser Value Was “046220006 for cisplatin. This suzzests that the ionic cisplatin complex has a notable affinity for the aqueous phase of hydrophilic environments. It is important for cisplatin to be inctuded in the inner phase of the wonanoemubions. On the other hand, carboplatin showed a logPoiaer value of -0.232(.07. Carboplatin is produced by substituting the chlorides in Gsplatin with the I,L-cyclobutane dicarboxylate ligand. ‘Hence, carboplatin cannot form an aqueous complex, This drug demonstrated less partitioning in the aqueous phase, resulting in less encapsulation in the inner phase of the nanoemulsions. The closeness of the release behavior of the ‘wo drugs indicates the negligible influence ofthis lower level of encapsulation, ‘The drug released from the inner phase supplements «drug depleted in the extemal phase, supplying sustained and controlled release as was observed by the in vitro cisplatin release behavior. This is reasonable since nanoemulsions are considered to be prolonged drug release systems with the oil and emulsifier layers acting as release barriers, Sustained delivery via an intravesical route can ensure the continuous presence of drug in the bladder. It is plausible to expect an increase in efficacy with increased curation of direct contact between the drug and urothelium (7). The release rate generally increases with smaller drop- lets, since a smallcroplet system has a larger total surface area through which drug diffusion can oocur (12.31). Thismay have contributed to the higher release rate of Brij 98- containing nanoemulsions (N2 to N4) than the Tween §0- containing system (NI). The viscosity of the nanoemulsions is another important factor inthe drug release characteristics. It js recognized that increasing the Viscosity of wo emulsions causes a more rigid structure and decreases the rate of drug release (11,32) There was a correlation between the viscosity and the amount of drug released by the four nnoemulsions tested, suggesting a role of viscosity in cispatin release from these gysiems. The addition of okic acid (NB) retarded drug release from the carriers This may have been due to the ability of oleic acid to be firmly anchored at the level of the ‘emulsifier film (3), thus strengthening the interfaces among droplets. It Was apparent that the urine reduced cisplatin release for most cariers testecL Since intravesical transport can be adversely affected by dilution of the instilled drug vehicle by urine in the bladder (7), the dilution by urine Fed to a reduction in the concentration of cisplatin and subsequent drug release, Nanoemulions with Brij 98 (N2 to N4) showed a greater reduction in cisplatin release compared to the 2321 carrier with Tween 80 (NL), Itis possible that wio emulsions can be comerted into waterin-oilsn-water (wialw) multiple emulsions upon dilution with water @5}. In wo ‘emulsions, the oil phase can be reganied a a semipemeable ‘membrane allowing water transport. Water flux into the inner phase of the systems may then be induced by an osmotic gradient (4). The release of the drug from the droplets is reduced when water enters the aqueous phase and preduces ‘an osmotic (Swelling) effect. The downregulation due to ‘swelling can be explained by a reverse solvent drag or by a reduced dig, concentration gradient after swelling occurs ‘Emuions with small droplets are more responsive to osmotic pressure than these with larger droplets (3435). Our results are in acoordance with this theory, “The antitumor effect of cisplatin depends on the concen- tration and exposure time (36). Cisplatin and carboplatin in ‘aqueous solution produced a burst of accumulation in bladder tissues after the frst sampling (I h). The drug concentration ‘was further reduced following an increase in the administra tion duration, This may haye been due to dilution of the drug by urine, The concentration gradient for promoting drug diffusion into the blackler was thus decreased (6). This result ‘was expectedt since the duration of drug instillation during intravesical blacker therapy is typically limited to 2 h (15). ‘The urothelium is the toughest known barier to drug celivery G7). The main ste of bladder permeability is located at the superficial cell in the epithelia called umbrella cells These barrier functions may prevent drug permeation into the bladder wall, especially hydrophilic drugs Free carbopl- tin showed higher permeation compared to free cisplatin because of the less hycrophilic characteristic of carboplatin. ‘The én vivo results indicate that nancemulsions increased cisplatin deposition in bladder tisste, except the formulation with Tween 80 (ND. Factors contributing to the increased permeation by the nanoemulsions may be the large surface area cue to the small croplet size. The availability of the drug is enhanced because the small sia: improves contact with the surface. Wetting and spreading can be enhanced by the low surface tension of the nanoemulions (38). Due to the higher cisplatin concentration gradient on the blacker surface, the drug molecules are efficiently transported towards the ‘urothelium from the nanoemulsions. Another possible mec= anisms that the acid pH can increase the ion permeability of the urothelium (13). The pH of the nanoemulsions was around 5, as summarized in Table I However, this cannot explain the low drug transport of the Tween SO-containing formulation (NI). ‘The efficiency of drug absorption is increased with formulations possessing bioadhesive characteristics (7). Vise cosity can be an index of the adherenae properties. Since the Table Tl The characterization of waterdnoil nanoemulsions by ‘oplet si, zeta potential and pH Coke Zeta potential (mV) pH NI 3208 38 x2 M404 56 NB 35103 52 NE “47203 Sa ‘Each value represents the mean=SD (1=3).232 NI formulation showed the highest viscosity, the muooadhe- sive property might not have been the controlling factor in intravesical cisplatin delivery. Nanoemulsions with Tween 80 (NI) exhibited slower drug release. Although the carriers with sustained-release activity can serve as drug. depots, thereby extending the drug exposure in the bladder cavity after the voiding, of urine, the extremely slow drug release may lead to a failure to provide sufficient cruz molecules to the target tissues. This suggests the importance of the formulation design for intravesical delivery after completely considering the physicochemical characteristics and release behavior ‘Although N2 and N4 showed increased! cisplatin uptake in the bladder wall compared to the free control, this enhancement only persisted for 2h. Afier that time, a lange quantity of urine may have infused into the cavity. The loading of a-terpineol (N3) could overcome this limitation to prolong a high cisplatin accumulation for a 3-h duration. This formulation even provided a greater carboplatin permeation than the free control until the end of the experiment (4 h), Approaches to enhance urothelium permeability by enhancers were investigated in previous research Chitosan, polyearbophil, protamine sulphate, and hyaluronidase are examples of permeation enhancers which can promote intrae vesical delivery (5.133940). Enhancers cause desquamation of the urothelium, which results in disruption of the diffusion barriers, including the glyoosaminoglycan layer, membrane plaques, and tight junctions of umbrella cells. Consequently, increased amounts of the drug can permeate into the Walle a- ‘Terpineo! has long been used as an enhancer for skin delivery because of its ability to disrupt intercellular lipid bilayers and its noneiritative nature toward the skin structure (41,42). The lipophilic aterpineol can easily partition into the oil phase of nanoemuions, The CLSM results confirmed the enhanced permeability of the nanoemulsion with c-terpineol (N3), This incorporation not only enhanced the absorption amount but also increased the depth of permeation as observed in the fluorescence images. Some detachment of the bladder surface observed in the histological examination may have reduced the barrier function, thus making the urothelium more permeable to cisplatin and carboplatin, Sustained intravesical delivery of drugs ata high level can guarantee the continuous presence of the drug in the bladder without requiring intermittent catheterization. Hence, increased efficacy of antitumor axtivity woukl be expected. This enhancement ‘was not detected by adding oleic acid, which is also a skin permeation enhancer. ‘One immediate insilation after TUR is recommended in all patients. In lowerise patients, no further treatment is needed before recurrence (43). One insillation of the nano- emulsions for a 3h chwell time was performed to examine the preliminary safety to bladder tisues. The safety of the Brij S8-oontaining system without enhancers (N2) was confirmed because of the evidence from histological observations, Some detachment of the superficial tissues treated with the nano- emulsion containing c-terpineol (N3) may have reduced the barrier property of the urothelium. This is an important mechanism of the enhancing potential of N3 to deliver the drug into the urothelium and the underlying tissue, The disrupted urothelium was more permeable than normal urothelium, This disruption was partial and possibly rever- ‘Hwang, Fang, Chen and Fang sible in the clinical status because of the revivifcation of the ‘membrane, Further investigation, such as recovery duration, of the bladder wall, is needed to elucidate the safety concem, of the nanoemulsions. CONCLUSIONS With increasing interest in intravesical chemotherapy, .wio nanoemulsions may prove to be promising new camiers for cisplatin and carboplatin. Cisplatin nanoemulsions showed. significant activity against bladder cancer cells. The int vitro study showed that the nanoemulsions procluced sustained, release of the drugs. The in vivo study using the intravesical, delivery of nanoemulsions with Brij 98 indicated that a high. concentration of the drug coukl be maintained in bladder tissue for at least 2h, Among all nanoemulsions examined, the ceterpincobcontaining system may be the most suited for future development because it provided controlled drug, rekase, high cytotoxicity. and high drug permeation into bladder tissues The high drug accumulation within the bladder wall coukl be prolonged for as long as 3~4 h with, this formulation. This é especially important for intravesical drug instillation Since the divell time is generally limited toh because of urine proxluction. The continuous presence of drug, in the bladder by sustained release, the increased availability of the drug contacting with urothelium by the small size ofthe systems, Wetting and spreading, because of the low surface tension, acidic pH environment of nanoemulsions, and. degyuamation by the enhancer may contribute to the high retention ability of cisplatin/carboplatin in the bladder wall. The great cytotoxicity against cancer cells by cisplatindoaded, nanoemulsions further encourages the use of the nano emulsions for intravesical instillation to treat tumors, Nano- ‘emulsions hold promise for the future of intravesical delivery of drugs into the bladder. REFERENCES 1, Nagabhushan TL, Maneval DC, Benedict WE Wen SE Ihnat PM, [Engle H, ef at. Enemoament of inirvestal delnery with Syn3 polenta inferferontphed gene thezpy for superic bladder CGmcet. Cytokine Growth Factor Rev. 200718:309-334, 2 Avrischer EB, Cooksley CD, Grossman HB, Sabichi AL, Hambin L Diney Ca of Cina nolo tie oto ‘GIncer and associated complications. 7 SI0-553 3. Wits JA, Hendricksen K. Inuawvesical pharmacotherapy for rontmusckeimasive blakler canoer a critical analysis of cur renlly available dues, treatment schedules, and long-term esulls Eur Urol, 2008334552. 4 Skinner E, Willa new navesical chemotherapy agent improve the teatment of nonemuscosmaasve bladder cangax? Nat Cha act Urol, 20074248249, S. Shen Z, Shen T, Wiemfjes MG, O'Donnell MA, Au JLS. Inirivsical ueamens of bladder cancer review. Pharm. Res. 20OR51STD-1510. 6 LUZ, Yeh TK, Tsii M, AWJLS, Wientjes MG. Paditaxebioaded gelatin nanopartides for intrrescal blakler cmcer therapy Gin Cancer Res. 2005107677764, 7. Tyagi Wu PC. Chancellor M, Yoshimura N, Fang L. Recent advances in intavesical druggene delivery. Mol Pharmaceut. Dune O57, 8 Pectasides D, Pectasifes M, Boonamopoulos"T Sistemi chemo- therapy in Joaily achanced andicr metagatic badder enoct, (Cancer Treat Rev. 2006S.Permeation Enbancer-Containing Watern-Oil Nanoemulsions as Carriers for Intravesical Cplatin Delivery 9. Saxena S, Agrawal U, Agarwal A, Murthy NS, Mohanty K. Adwantingavescl therapy tesed cn an. vito GrOUNC, asa nthe management of sete transitnal el ence of tho urinay Hae 5 Ine 1012-1017. Hiadachik BA, ter Borg Mt Jada J, Svery RD, So Al, Burt FM, ot Poca ad explains raves agents {eden onload ce BIC SS Wu H, Ramachandran C, Bielinska AU, Kingzett Ky Sun Ry ‘Weer ND. era Topral transection BNAina aterincl tnt) Pha ADL 3 Wang tl Hurg cB Yoh CH Ring JV Tie rlease analgesic cs of morptine and fs Gr prod mown propor a, frmulated by water Ranaemusens. J Dri Tae, Zansteaecl. Racha Me Nev tonta an imraesiad eps an che sr fronts ina diver. Eur Urol ore SHH9 ‘irdvnam Fa Person S Hunan unc chemical compastin sou inti. Nir el Ass, DHA Burns Gastin J, mince Choz JE. ty fnirnesial chemthfap, ur Urol 20065095 ‘Wang Jp Maint Y, Takayama K. Aitimor effets and areca of acho A rl by etic impart of wenn Fs Gist and PEG ip Sharm Soros a ‘Hung CE, Fang CL, Liao MH, Fang JY. The effect of oil and dg dlvery tastes bl eu wy ps cen. I) Pars Suorsss9-200, Hung CR, Chen JIG Lito MH, Lo HM, Fang JY, Development and evaluation of emusiomfiposome” blends for reseratol Selvey. J Nanossi Nancrectnol 0629S. Hurst RE, Zebrovsk Re Identiti of proteoglycans present high dey on tonne snd urman blader hum sflace 3 Urol SKISI6HAL6sL Lawrence Mi, Recs GD. Microcmuisics-based media as novel drug delivery gstems Adv Dig Deliv Rev. 20004585-121. 21. Steaniak M, Nest) A, Kuch J. lick J, Pyka A. Use of RISTLC to dbtermine the by values of Somers of orgie campauncs Chrematceriphi. 02579. ‘Buse K, Hamsch $, Miller RH, Muller BW-The inluence of alll fatty acl on th: Properties and the sabity of parenteral ‘lw emulsions mexdiicd with Solulol SIS". Eur J) Pharm Bioplamn 2eRi35139. Solins C, ingicréo B. Nella J, Azcmar N. GarsisCelna Mi Nanoumlsonn. Curr Opn Co Interace Se 2005 ED-L0. ‘Avannies CA. Kits G, A theological sly on microemuion gob of Bepropyl myrisate, pobsorbate sO, ulyeerok and wate. J Cosmet So. 193Reir1=7. Kars HY, Kantulut B, Goker E, Ginesi T, Gabor Cantolled reiease of metorexate tram wo microsnukion and its vioo antitumor activity. Drug Deli 204 225-983 Hawes A,’Amsden B. Carprotiedn delivery methods, Pam Res AISI 27, Fang 1% Hung CR, Hua SC, Hoang TL, Acustcally active peMborccarton nanoemubions a6 drug ceiNery Guts for 0, B Mw 15, ctfcagy of 16. i 1 1B mn, * a. 8 0. 4k. cs 233 Ccamptothocin: drug release and cytoenicity against cancer cel. Ulrasonics, 2005496, ‘Kim JH, Kim_ YS, Paik K, Lee S, Nam HY, Min KEL at Antitumor efficacy of cislatinebaded slyco} chitosan: nano- artes in tnoebeanng mis J'Cone Rela 3m 27 410. ‘orsuyenagi T, Usami M, Nod Y, Nagata M, Computational consideration Of Gsplatin indfolyss and acid dissociation in ‘aqueous malic effet of total drug concentrations. Int J Pharm 2D 495104 Hang TL Tee WR, Hu SC Fang J¥ Cepatin encarta pphosphatidylethanolamine Epesomes enhanocs the it Vio Gylotexicty and i vio intratumor drug accumulation agains. ‘melanomas, J Dermatol So, 20074¢c1!—2, Gung Fi, Kim TW, Kwon M, Kwon IC, Jeong SY. Oil characteristis and function of ‘drug or gene delivery sysem. J ren Sera as coy pea SER AnG MRR C Far tiscnn Samco a eine ected el ent ees ‘Bjerregaard S, Séderberg I, Vermehren C, Frokjaer S. Formus Ei saree crac enter Ioan Ss 8 a 'S, Sddlerberg L, Vermehren C, Frokjaer S. The effect ‘ron sanots ate on rebase and owe properties of iuacig nye one Konishi M, Tabata, Kariya M, Hosseinkhani H, Suaiki A: Eee Lani ore Se psy Karn tr 8 Kcte model et dru dstobuton i the unnay bad wall apc) Ras Heo Sha hindu any be Lee rca cee Paved peat of abut ro ie srrplatalte (tems dt as thou adr wa 9 Pr. 3cLO™= 73 oe ce ee Eon wae aaron ERE See neta terranes rar pect ean Be De Ta Beste pas mac tong mokeular dynamics of an spin label in stratum: iitson ames oti pets eis ina a Shisdoimashe thier camera ssemide novo ce ie See eae acre SSRReproduced with permission of the copyrightowner. Further reproduction prohibited without permission.