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submicron‐emulsion systems
Intravesical Delivery of 5-Aminolevulinic Acid from Water-in-Oil Nano/Submicron-Emulsion Systems JIA-YOU FANG,1 PAO-CHU WU,2 CHIA-LANG FANG,3 CHAO-HUANG CHEN1 1 Pharmaceutics Laboratory, Graduate Institute of Natural Products, Chang Gung University, 259 Wen-Hwa 1st Road, Kweishan, Taoyuan, Taiwan 2
Faculty of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan
3
Department of Pathology, College of Medicine, Taipei Medical University, Taipei, Taiwan
Received 31 March 2009; revised 27 September 2009; accepted 29 September 2009 Published online 17 November 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.22006 ABSTRACT: The present work reports on the development of water-in-oil (w/o) emulsions for the intravesical administration of 5-aminolevulinic acid (ALA). The physicochemical properties of droplet size, zeta potential, and viscosity of the emulsions are characterized and the ability of the emulsions to release ALA following in vitro application is tested. The delivery systems are administered intravesically for 1 and 3 h in rats to examine the drug accumulation in bladder tissue. The mean size and zeta potential of the emulsions are 50–200 nm and 3 to 14 mV, respectively. The loading of ALA into the emulsions resulted in a slower and sustained release. The release extent was found to be inversely related to the droplet size of the emulsions. The emulsions did not increase the drug permeation into tissues during short exposure duration (1 h). When the dwell time was extended to 3 h, the systems showed a 2.7-fold increase in the ALA concentration in the bladder wall. Images of confocal laser scanning microscopy demonstrated a higher and deeper fluorescence signal, with emulsion administration, as compared to the aqueous control. Intravesical emulsion delivery provides a significant advantage for drugs targeting bladder tissues. ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:2375–2385, 2010
Keywords:
emulsion; 5-aminolevulinic acid; intravesical delivery; bladder; sustained release
INTRODUCTION Bladder cancer is the fourth most common malignancy among men in Western countries, and the high recurrence rate makes it the most prevalent malignancy.1 A standard treatment for patients with bladder cancer is surgical transurethral resection (TUR) of the tumor. Intravesical chemotherapy is widely used as adjuvant therapy after TUR, to reduce the risk of recurrence.2 The intravesical route permits site-specific delivery of drugs, with a reduced side-effect profile compared to oral delivery systems, either by avoiding first-pass metabolism or by obtaining a local effect.3 Intravesical delivery is an administration method of inserting a catheter from the urinary tract to the bladder cavity to deliver drugs. 5-Aminolevulinic acid (ALA) is a precursor of protoporphyrin IX (PpIX), which is used in photoCorrespondence to: Jia-You Fang (Telephone: þ886-3-2118800 ext 5521; Fax: þ886-3-2118236; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 99, 2375–2385 (2010) ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association
diagnosis and photodynamic therapies (PDT) of malignant bladder cancer.4,5 It combines photosensitizers that selectively bind to tumor tissues and a powerful intravesical light source to destroy the complex formed by the tumor cell and photosensitizer.1 ALA can reduce the recurrence rate in stage Ta/ T1 bladder transitional cell carcinoma.6 It also helps to detect the location of bladder tumor for clinical use.7 ALA is a hydrophilic molecule with a molecular weight of 167.7. The efficacy of intravesical therapy for bladder cancer is in part limited by the poor penetration of hydrophilic drugs into the urothelium.8 The half-life of ALA in the body is quite short (45 min) due to its insufficient stability under physiological conditions.9 Nanosystems with a welldefined particle size and shape have immense potential for intravesical delivery as they can enhance the ability of drugs to cross the urothelium and prolong drug retention near the tumor site.10 Moreover, the encapsulation of drugs into nanosystems can protect the molecules, and thus, prevent instability. Emulsions stabilized with nonionic
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surfactants are a class of nanosystems with a droplet size of 20–200 nm.11 Water-in-oil (w/o) nanoemulsions are considered to be attractive prolonged release systems for hydrophilic drugs, with the oil and interfacial layers acting as a release barrier.12,13 The aim of this study was to develop w/o emulsions as intravesical delivery systems to deliver ALA to the bladder for antitumor therapy. The mean droplet size, surface charges, and viscosity were evaluated to determine the physicochemical characteristics of the emulsions. The drug release properties of the emulsions were determined in vitro using a Franz cell. ALA permeation into the bladder wall after intravesical administration was examined using rats as an animal model. The possible disruption of normal bladder tissue by these w/o emulsions was also investigated for safety reasons.
of the emulsions are shown in Table 1. Under the processing temperature (508C), ALA molecules could remain stable without degradation by immersing a 0.1% ALA in double-distilled water at 508C for 30 min. The amount of ALA in water before and after the heating was quantified by HPLC. Size and Zeta Potential of Emulsions The mean size (z-average) and zeta potential of the emulsions were measured by photon correlation spectroscopy (Malvern Nano ZS1 90, Worcestershire, UK), using a helium–neon laser with a wavelength of 633 nm. The formulations were diluted fivefold with sesame oil before measurement. Photon correlations of spectroscopic measurements were carried out at a scattering angle of 908. The stability of emulsions was determined by monitoring size and zeta potential at 378C and 65% relative humidity for 28 days.
MATERIALS AND METHODS
Viscosity of Emulsions
Materials
The viscosity of the emulsions was determined using a programmable viscometer (DV-IIþ, Brookfield, Middleboro, MA). The spindle (s21, 170 mm) was rotated in the samples, and viscosity was recorded 60 s after rotation commenced, when the level had stabilized. The speed of the paddle was set to 30 rpm.
5-Aminolevulinic acid (ALA), sesame oil, and Span 80 were purchased from Sigma Chemical (St. Louis, MO). Tween 80 was obtained from Kanto Chemical (Tokyo, Japan). Plurol diisostearique (Plurol) was obtained from Gattefosse´ (Gennevilliers, France). Brij 98 was provided by Acros Organics (Geel, Belgium). Hydrogenated soybean phosphatidylcholine (SPC, Phospholipon1 80H) was supplied by the American Lecithin Company (Oxford, CT). Preparation of w/o Emulsions The oil and aqueous phases were separately prepared. The oil phase consisted of oil and surfactants, while the aqueous phase consisted of double-distilled water and the drug (to achieve a concentration of 0.1% (w/v) in the final product). The two phases were heated separately to 508C. The oil phase was further mixed using a high-shear homogenizer (Pro250, Pro Scientific, Monroe, CT) at 12,000 rpm for 10 min. The diameter of the generator was 5 mm. Then, the oil phase was added to the aqueous phase and sonicated using a probe-type sonicator (VCX600, Sonics and Materials, Newtown, CT) for 10 min at 35 W. The total volume of the final product was 10 mL. Compositions
In Vitro ALA Release ALA release from the w/o emulsions was measured using a Franz diffusion cell. A cellulose membrane (CelluSep1 T3, with a molecular weight cutoff of 3500) was mounted between the donor and receptor compartments. The donor medium consisted of 0.5 mL of vehicle containing ALA. The receptor medium (5.5 mL) consisted of pH 6 citrate-phosphate buffer. The available diffusion area between the cells was 0.785 cm2. The stirring rate and temperature were set to 600 rpm and 378C, respectively. At appropriate intervals, 300-mL aliquots of the receptor medium were withdrawn, and then immediately replaced with an equal volume of fresh buffer. The amount of ALA released was determined by highperformance liquid chromatography (HPLC). In the study examining the influence of urine on ALA release, 0.5 mL of synthetic urine was added to the
Table 1. The Compositions and Their Percentages (%, v/v) of Water-in-Oil Emulsions Used in This Study Code E1 E2 E3 E4
Water
Sesame Oil
Span 80
Tween 80
Plurol
Brij 98
SPC
10 10 10 10
44 44 44 44
30 — 30 28
16 16 — 16
— 30 — —
— — 16 —
— — — 2
SPC, soybean phosphatidylcholine. The hydrophile–lipophile balance (HLB) of Span 80, Tween 80, Plurol, and Brij 98 is 4.3, 15.0, 4.5, and 15.3, respectively. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010
DOI 10.1002/jps
DELIVERY OF ALA FROM W/O NANO/SUBMICRON-EMULSION SYSTEMS
donor compartment. The synthetic urine solution contained 14.1 g NaCl, 2.8 g KCl, 17.3 g urea, 1.9 mL of a 25% (v/v) ammonia solution, 0.6 g CaCl2, and 0.43 g of MgSO4 made up to 1 L with water.14 HPLC Analysis of ALA The fluorescence derivation of ALA samples was based on a modification of the Hantzsch reaction.15 The ALA contents of samples were analyzed by HPLC, as cited previously.16 Cytotoxicity Assay
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PpIX in the bladder tissue was extracted by a homogenization method. One milliliter of a 1.5 M perchloric acid/methanol (1:1, v/v) mixture was added to the excised tissue in a glass homogenizer and ground for 5 min. The resulting solution was centrifuged for 10 min at 10000 rpm. The fluorescence of the supernatant was determined spectrofluorometrically (Hitachi F-2500, Tokyo, Japan) at an excitation wavelength of 403 nm and emission at 602 nm. Confocal Laser Scanning Microscopy (CLSM) Bladder samples obtained following in vivo instillation were examined for PpIX fluorescence images by CLSM. The thickness of the bladder wall was optically scanned at 10-mm increments through the Z-axis of a Leica TCS SP2 confocal microscope (Wetzlar, Germany). Optical excitation was carried out with a 543-nm He–Ne laser beam, and fluorescence emission was detected at 560–620 nm.
Human urothelial carcinoma cell line (T24) was purchased from the American Type Culture Collection (Rockville, MD). The cells were seeded at an initial concentration of 1 105 cells/well in a 24-well plate, and incubated in a 1 mL medium (10% fetal bovine serum, 89% RPMI 1640, and 1% penicillin– streptomycin). Water and emulsions, with or without ALA diluted with medium, was added at 24 h postinoculation, and plates were incubated in a 5% CO2 atmosphere at 378C. After 4 h of incubation, each plate was then exposed to light from a tungstenhalogen lamp (Osram Decostar51, 50 W) for 40 min. The total spectral irradiance at the level of the cells was 10 mW/cm2, as measured with a light meter (illumination meter). Subsequently the medium was continuously incubated for 72 h. After PBS washing, cells were incubated with 5 mg/mL 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) in RPMI 1640 for 2 h at 378C. Formozan crystals resulting from MTT reduction were dissolved by adding 200 mL DMSO and gently agitated for 30 min. The absorbance of supernatant was then measured spectrophotometically in an ELISA reader (Fluostar Optima, BMG Labtech GmbH, Offenburg, Germany) at 550 nm. Cell viability was calculated as the percentage of the control.
RESULTS
In Vivo Intravesical Instillation into Bladder Tissues
Physicochemical Characterization of the Emulsions
Female Sprague–Dawley rats, weighing 225–250 g, were used in this study. The animal protocol was approved by the Institutional Animal Care and Use Committee of Chang Gung University. Animals were anesthetized with urethane (1.2 g/kg) subcutaneously before intravesical instillation. The residual urine was evacuated by pressing the lower abdomen. A polyurethane catheter (BD Angiocath Plus1, Becton Dickinson Korea, Gyeongbuk, Korea) was inserted into the bladder. The bladder was washed with 0.5 mL normal saline. Subsequently 0.2 mL of emulsions was instilled into the bladder. The catheter was removed and the urethra was ligated with cotton thread. The dwell time for instillation was 1 and 3 h. After the application time, the bladder was excised and washed with saline.
The droplet size of the w/o emulsions ranged 50–200 nm, as shown in Table 2, which fall in the range of so-called nanoemulsions (20–200 nm). For the strict definition of nanotechnology, a size of <100 nm is feasible for qualification as a nanosystem. Hence the final products prepared in this work are nano- or submicron-sized emulsions. There was a difference in the average size of the droplets, determined according to the types of emulsifier used. The standard formulation (E1) was stabilized by a combination of two surfactants: a sorbitan mono-9octadecenoate (Span 80) and a polyoxyethylene 20 sorbitan monooleate (Tween 80). The size analysis indicated that this nanoemulsion had a mean droplet size of 108 nm. Span 80 with a hydrophile–lipophile balance (HLB) of 4.3 was replaced with Plurol
DOI 10.1002/jps
Histological Examination of Bladder Tissues Immediately after treatment with an aqueous solution, or the w/o emulsions by intravesical instillation for 3 h, a specimen of the exposed area was taken for histological examination. Each specimen was fixed in a 10% buffered formaldehyde solution (pH 7.4) for at least 48 h, and stained with hematoxylin and eosin. For each sample, three different sites were examined and evaluated under light microscopy (Eclipse 4000, Nikon, Tokyo, Japan). Statistical Analysis Statistical analysis of differences between different treatments was performed using unpaired Student’s t-test, and a 0.05 level of probability was taken as the level of significance.
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Table 2. The Characterization of Water-In-Oil Nanoemulsions by Droplet Size and Zeta Potential
Code
Size (nm) at Day 0
Size (nm) at Day 28
Zeta Potential (mV) at Day 0
Zeta Potential (mV) at Day 28
Viscosity (mP s)
pH
E1 E2 E3 E4
108.3 10.7 194.3 18.2 50.4 2.9 209.9 9.6
86.6 8.6 176.6 3.7 38.0 0.5 170.5 10.0
7.4 0.6 10.0 0.7 2.5 0.2 13.6 0.9
10.3 2.6 8.7 0.6 1.6 0.7 10.2 2.4
105.2 5.0 204.2 1.8 188.7 11.9 262.7 11.1
5.2 7.0 4.9 6.0
Each value represents the mean SD (n ¼ 3).
(polyglyceryl diisostearate), with an HLB of 4.5 in the emulsions (E2). This substitution led to an increase in size to 194 nm ( p < 0.05). Brij 98, another hydrophilic emulsifier with an HLB of 15.3, was substituted for Tween 80 (with an HLB of 15.0) to produce an emulsion (E3) with a reduced size of 50 nm ( p < 0.05). SPC was also incorporated into the w/o interface of emulsions as a coemulsifier (E4). Results indicated that a significant enlargement in size (210 nm) occurred after SPC incorporation (E1 vs. E4, p < 0.05). As depicted in Table 2, the zeta potentials of the emulsions ranged from 2.5 to 13.6 mV. SPCcontaining emulsions (E4), which showed the highest zeta potential, as compared to other systems ( p < 0.05). The addition of Plurol resulted in a slight but significant increase (E1 vs. E2, p < 0.05) in surface charge. On the other hand, Brij 98 shielded the zeta potential (E1 vs. E3, p < 0.05). The local retention condition of tissue is an important parameter for drug delivery by emulsions (11). The viscosity/rheological properties are factors affecting retention ability. As summarized in Table 2, the value of viscosity depends on the ingredients used in the emulsion formulations. The respective replacement of Span 80 and Tween 80 (E1) by Plurol (E2) and Brij 98 (E3) increased the viscosity 2-fold. Adding SPC to the systems (E4) further increased the viscosity 2.5fold. This may indicate that Plurol, Brij 98, and SPC themselves possessed a greater viscosity as compared to Span 80 and Tween 80. After 28 days of storage at 378C, the mean diameter of E1 and E2 was maintained (Tab. 2). The presence of Brij 98 and SPC (E3 and E4) slightly, but significantly, decreased ( p < 0.05) the size after storage. Concerning the zeta potential, there were no or negligible changes observed for all formulations from storage.
and continuous release. The release of ALA from the emulsions followed the Higuchi diffusion controlled model equation (release percentage: h1/2). The result showed that the ingredients are important for modulating ALA release. The substitution of Span 80 (E1) by Plurol (E2) further slowed the ALA release. The same phenomenon was observed by incorporating SPC (E4) into the systems. Only 6% of the drug dose had been released from the donor compartment after a 12-h application. Emulsion systems, as prepared with Span 80 and Brij 98 (E3), produced a 2.6-fold enhancement in the
In Vitro ALA Release The in vitro release behavior of ALA is shown in Figure 1A. Release of ALA from double-distilled water was studied as a control group. As can clearly be seen in Figure 1A, ALA release from the water was rapid, with 60% released after 4 h, whereas, the release behavior from the w/o emulsions exhibited a slower JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010
Figure 1. In vitro release percentage (%) of 5-aminolevulinic acid across a cellulose membrane from double-distilled water (control) and emulsion systems (A) and in the presence of synthetic urine (0.5 mL) in the donor compartment (B). All data are presented as the mean SD of four experiments. DOI 10.1002/jps
DELIVERY OF ALA FROM W/O NANO/SUBMICRON-EMULSION SYSTEMS
released amount of ALA, with respect to the standard emulsion (E1). Synthetic urine was added to the donor phase and mixed with the emulsions in order to simulate in vivo conditions of urine infused into the bladder. ALA release in the presence of urine is demonstrated in Figure 1B. The percentage of released ALA from the aqueous solution (control) was reduced in the presence of synthetic urine, possibly due to the dilution effect. The standard emulsion (E1), and the systems with Brij 98 (E3) also showed decreased ALA release after loading with urine. This reduction was negligible in the formulations with Plurol and SPC (E2 and E4, p > 0.05). Cytotoxicity Assay To assess the cytotoxicity of ALA-loaded emulsions, their tumor-killing activity was determined against an urothelium cancer cell lining by MTT assay. The effect of light exposure on the viability of cells by free ALA solution was first examined, as presented in Figure 2A. The PDT assembly was set according to
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the previous study.17 Cells exposed to ALA but no light showed a limited decrease in cell death. The results of survival after PDT of the cells exhibited that the light is essential to induce proliferation inhibition. All further experiments were performed under PDT treatment. As shown in Figure 2B, the emulsions with Brij 98 (E3) completely inhibited the growth of urothelial carcinoma cells in both doses (0.3 and 0.5 mM). The standard system (E1) also showed a significant cytotoxicity to the cells. Antiproliferative activity was negligible ( p > 0.05) for the vehicle with Plurol (E2) at a dose of 0.3 mM. The incorporation of SPC in the system (E4) led to the attenuation of the antiproliferative activity of the emulsions. Generally, the cytotoxicity increased in a trend of E3 > E1 > E4 > E2. Empty nanosystems, without ALA, were also tested, and it was found that the vehicles themselves had no effect on cytotoxicity. This indicates that the cytotoxicity toward the urothelial carcinoma cells was mainly a consequence of the ALA molecules. In Vivo Intravesical Instillation into Bladder Tissues Rats received an intravesical dose of ALA to examine the drug amounts in the bladder wall after 1- and 3-h applications. The emulsions with Plurol (E2) were not included in this experiment because of the extremely low ALA release and cytotoxicity from this emulsion. ALA is converted to PpIX after permeation into the bladder wall. As shown in Figure 3, PpIX accumulation from the aqueous control was 1.22 and 0.54 nmol/g bladder tissue with 1- and 3-h dwell times, respectively. The accumulation at 1 h from w/o
Figure 2. Viability (%) of human urothelial carcinoma cell line (T24) following treatment with free ALA in water, with or without light exposure (A), and emulsion systems with light. (B) Each value represents the mean SD of five experiments. DOI 10.1002/jps
Figure 3. In vivo protoporphyrin IX (PpIX) accumulation (nmol/g) in bladder tissues after intravesical instillation of double-distilled water (control) and emulsions with 5-aminolevulinic acid (0.1%) for 1 and 3 h. All data are presented as the mean SD of six experiments. Significantly higher ( p < 0.05) as compared to the control at the same time plot; Significantly lower ( p < 0.05) as compared to the control at the same time plot. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010
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emulsions was only one-half of the accumulation from the control. There were no significant differences ( p > 0.05) among the accumulation levels of the three emulsions at 1 h. The PpIX concentration at 3 h showed a contrary result, as compared to that at 1 h. The standard emulsions (E1) produced a 2.7-fold enhancement in the average concentration of total PpIX in tissue sections. Changes in the hydrophilic emulsifier from Tween 80 to Brij 98 (E3) did not significantly change ( p > 0.05) the PpIX accumulation at 3 h. On the other hand, PpIX permeation decreased slightly, but significantly ( p < 0.05) when SPC was added to the formulations (E4). CLSM was utilized to observe the superficial PpIX distribution in the bladder wall. The superficial layer was optically scanned at 10-mm increments for nine fragments from the surface of the mucosa, as shown in Figure 4 (left to right, top to bottom). The total 90 mm comprises the urothelium and a part of the lamina propria. Figure 4 shows confocal images, as obtained from the controls and w/o emulsions after ALA instillation for 3 h. An increase in red fluorescence at 488-nm excitation and 590-nm emission was observed and considered to be PpIX accumulation. Compared to the control (Fig. 4A), the increase in red fluorescence in the superficial layer, as treated with emulsions (Fig. 4B and C), can clearly be seen with intense signals. Only a pale signal was detected in the first three images (30 mm) of the control group. The ALA loaded in the emulsions could have permeated the deeper layers. The emulsions with Brij 98 (E3, Fig. 4C) produced an even stronger signal than the standard formulations (E1, Fig. 4B), although there were no significant differences ( p > 0.05) between their PpIX accumulation levels extracted from bladder tissue. Histological Examination of Bladder Tissues A preliminary safety profile of emulsions on bladder tissue was evaluated by checking the histology, as depicted in Figure 5. Light microscopy indicated no observable damage to the whole bladder wall in the control group (Fig. 5A). It can be seen that the bladder wall is divided into three sections: the urothelium, which is not blood-perfused; the underlying lamina propria; and muscle layer of the bladder, which contains blood vessels and lymphatics. Photographs taken after staining the bladder treated with standard nanoemulsions (E1) revealed no adverse histological changes (Fig. 5B). The urothelium remained intact after a 3-h treatment with this system. When the bladder was treated with the instilled emulsions containing Brij 98 (E3), there was generally no significant change in the structures (Fig. 5C). However, it is noted that there was some JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010
Figure 4. Confocal laser scanning microscopic (CLSM) micrographs of rat bladder tissues after the in vivo intravesical instillation of 5-aminolevulinic acid (0.1%) for 3 h from (A) double-distilled water (control), (B) standard emulsions (E1), and (C) emulsions with Brij 98 (E3) (original magnification, 20). The specimen was viewed by CLSM at 10-mm increments through the Z-axis from the surface to the deeper layers (top to bottom).
detached superficial urothelium (Fig. 5D, depicted by green arrows). There was also slight degeneration of the urothelium (depicted by blue arrows). These changes were an early and mild disruption of the bladder structure. It can be concluded that the emulsions with Tween 80 (E1) showed a safer delivery to the bladder, as compared to that of Brij 98 (E3). DOI 10.1002/jps
DELIVERY OF ALA FROM W/O NANO/SUBMICRON-EMULSION SYSTEMS
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Figure 5. Histological examination of rat bladder tissues after in vivo intravesical instillation for 3 h with (A) double ¼ distilled water (control), (B) standard emulsions (E1), (C) emulsions with Brij 98 (E3), and (D) emulsions with Brij 98 (E3) in another view (original magnification, 200).
DISCUSSION Intravesical drug delivery continues to remain an ideal treatment option for therapy of superficial bladder cancer. However, conventional vehicles fail to provide sustained exposure or enhanced permeation of drugs to the urothelium.18 Emulsions have been widely explored as potential drug delivery systems. In the present work, nano- or submicronsized emulsions with an optimal formulation design provided controlled, sustained release of ALA. It was found that ALA permeation into the urothelium was enhanced by encapsulation into the emulsions, especially in the presence of urine in the bladder. The drug delivery potential of emulsions depends on the physicochemical properties of the droplets, such as the size, surface charge, and viscosity.19 Incorporation of Plurol (E2), instead of Span 80 (E1), led to significant increase in the droplet size. This may have been due to the spacious hydrophobic chains of Plurol, which have two stearic acid chains. The hydrophilic polyglycerol of Plurol was bound into the interface, whereas the fatty acid chains remained at the surface of the droplets. The droplet size can be increased following an increase in the interfacial film thickness. A similar phenomenon is shown in systems with SPC (E4), the structure of which possesses two alkyl chains. Brij 98 may have the ability to strengthen droplet integrity,20 resulting in the strong DOI 10.1002/jps
cohesion of the droplet and the avoidance of aggregation. Another possibility is the influence of HLB. The emulsifier system with a determined HLB may well have stabilized the emulsions which had a moderate polarity. The mixture of Span 80 and Brij 98 may facilitate a satisfactory emulsification for the whole system. Brij 98-containing systems (E3) were the only emulsion that was actually in the nano-scale (<100 nm). The physical stability of a nanosystem is one of the most important desired product characteristics. Emulsions developed in this study generally showed acceptable storage stability, after 28-day duration, although a slight decrease of the size was observed for E3 and E4. The aggregation or fusion of the droplets was always detected after storage, however, the size reduction was also possible from the destruction of micro- or nanoparticles, which is not uncommon during storage.21 Another possibility is the free fatty acids derived from sesame oil during storage. Fatty acids with some surfactant characteristics might reside in the w/o interfacial membranes, strengthening the emulsifying ability of the whole system, thus, reducing the droplet size. Further experimentation is necessary to confirm this hypothesis. Both Span 80 and Tween 80 are nonionic species. The negative charge in the interface is believed to be the result of the ionization of the free fatty acids derived from the hydrolysis of triglycerides in the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010
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sesame oil. Alkali fatty acids are thought to yield mixed films with a higher packing density of interfacial film-forming components.22 SPC in the interface of the emulsions (E4) was responsible for the higher negative surface charges. The major component of SPC (Phospholipon1 80H), as used in this study, was 80% phosphatidylcholine, which exhibits a net charge. The other components were phosphatidylserine, phosphatidic acid, phosphatidylglycerol, and phosphatidylinositol, all of which are negatively charged. These anionic fractions were responsible for the negative zeta potential of SPC-containing emulsions. It can be seen that the system with Plurol (E2) showed a higher negative charge as compared to that with Span 80 (E1). The structure of Plurol belongs to glycerides. The free stearic acids derived from Plurol may contribute to the negative charges within the interface. Further investigation is needed to elucidate this inference. The inner phase with smaller size possesses limited surface area for the droplets. This leads to the lower negative charge of the emulsions. This can explain the lowest zeta potential of the systems containing Brij 98 (E3) because of their smallest size. The viscous and elastic properties of the dispersions are important for their application in the bladder since they may provide a bio-adhesion to the bladder wall, thus prolonging the retention duration. The emulsions with SPC (E4) showed the highest viscosity. Moreover, both E2 and E4, with the larger droplet sizes, exhibited more-viscous properties, as compared to E1 and E3. This may have been due to the hydrophobic hydrocarbon tails of the SPC dangling out of the interface, which resulted in significant entanglements and enhanced viscosity.23 It is well established by experimental results that increasing the viscosity of the emulsion phase leads to an increase in the particle size.24,25 According to the generalized Stokes–Einstein equation developed by Mason,26 the hydrodynamic diameter of a droplet is inversely related to its viscosity. The contradiction may cause only a moderate correlation (Pearson’s correlation coefficient r ¼ 0.81, p ¼ 0.093) to be detected between viscosity and size. This suggests that, not only the size but also other factors, such as the emulsion structure and droplet-to-droplet interactions can influence nanoemulsion viscosity/rheology. For example, a higher number of smaller droplets results in more droplet-to-droplet interactions, and an increased resistance to flow. The main clinical limitation with ALA is its instability in its physiologic pH. ALA is relatively stable at lower pH values.7,27 The shelf-life of ALA, in a pH 7.53 aqueous solution, can be as short as 10 min.28 The inclusion of ALA in the inner phase of the emulsions may protect the molecules from degradation in tissues and urine. The acidic pH of the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010
emulsions, as shown in Table 2, supports good stability of ALA in these systems (E1, E3, and E4). This effect may produce prolonged drug exposure in the urothelium. In addition, the acidic pH increases the ion permeability of the urothelium.3 The release rate generally showed a trend of E3 > E1 > E4 > E2. The release kinetics of the Higuchi model assume that when one layer of w/o emulsion droplets is depleted of drug, release from the next layer begins.13,29 The drug is stably retained in the inner phase for a determined duration, followed by its slow release into the external phase. The oil phase may constitute an additional barrier, which prevented the partitioning of ALA into the continuous phase, thereby limiting the delivery. It is recognized that increasing the viscosity of w/o emulsions causes a more-rigid structure and decreases the rates of drug release.12,30 However, this did not occur in the present study since a low correlation (r ¼ 0.25, p ¼ 0.754) was calculated between the viscosity and released percentage at 12 h. The viscous properties of emulsions may increase the residence time at the mucosal membrane.18 According to the results of in vivo delivery, it seems that viscosity of emulsions is a negligible factor influencing ALA release and permeation. Another possible mechanism predominating ALA delivery is the influence of droplet size. The rate of release generally increases with smaller droplets, since a small-droplet system has a larger total surface area where drug diffusion can occur. Our profiles showed a good correlation (r ¼ 0.92, p ¼ 0.077) between the emulsion size and the released percentage. There is evident that the drug is more easily released with a small droplet size.25,31,32 Significantly reduced drugs released from the emulsions with Plurol (E2) and SPC (E3) were observed. The addition of fatty acid or ester with long alkyl chains, such as sodium stearate, is thought to yield a higher packing in the interfacial film.22,25 Both Plurol and SPC have additional alkyl chains, as compared to Span 80. It was apparent that urine reduced ALA release for most vehicles. A previous study10 suggested that intravesical transport can be adversely affected by dilution of the instilled drug solution by urine in the bladder. Responses to intravesical delivery are directly proportional to the drug concentration rather than the drug dose.18 Hence, the dilution by urine led to a reduction in the concentration of ALA and subsequent drug release. Nanoemulsions with smaller droplets (E1 and E3) showed a greater reduction in ALA release, as compared to the formulations with larger sizes (E2 and E4). It is possible that a water-inoil-in-water (w/o/w) multiple emulsion type was formed by mixing the w/o emulsions with water (urine). In w/o emulsions, the oil phase may be regarded as a semipermeable membrane allowing the DOI 10.1002/jps
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transport of water, and this flux of water into the inner phase of the emulsions may then be induced by an osmotic gradient.29 The release of the drug from the droplets is reduced when water enters the aqueous phase and produces an osmotic (swelling) effect. This osmotic downregulation of release has been reported in the case of w/o/w systems.29,33 The swelling downregulation is explained by a reverse solvent drag or by a reduced drug concentration gradient after the swelling of the inner phase. The emulsion with small droplets is more responsive to osmotic pressure than are larger droplets.29,34 The results of the present study are consistent with this theory. The cytotoxicity results showed that free ALA in water induced growth inhibition in the cells and ALA was slowly released from the emulsion droplets. A higher release rate may render a high level of free ALA for cell uptake. It is noticeable that E3 showed even higher antiproliferative activity than the free form ( p < 0.05) at a lower dose (0.3 mM). Hence, the activity against cancer cells might not have completely been due to the direct penetration of free ALA into the cytoplasm. Nanoparticles need to enter cells and diffuse through the viscous cytosol in order to access the particular cytoplasmic targets where the sites of action are located.35 Cellular uptake of the drug can possibly 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.36 Detailed investigation of the emulsion effect on cytotoxicity is not the main focus of this study. The basic mechanism is to be elucidated in future studies. ALA in aqueous solution (control) produced a higher PpIX accumulation ( p < 0.05) in the bladder wall after a 1-h administration due to the burst release of ALA, as confirmed by the in vitro release experiments. However, the accumulation was greatly diminished when the application duration was prolonged to 3 h because of the depletion of the drug as it was released. The duration of drug instillation during intravesical bladder therapy is typically limited to 2 h, after which time urine production is largely responsible for decreasing the drug concentration in the bladder.4,37 The bladder wall is composed of an unperfused transitional epithelium called the urothelium, which represents the toughest known barrier to drug delivery.10,38 Although the emulsions could not improve ALA permeation at the initial stage of application (1 h) because of the slow release from the inner phase, PpIX accumulation was significantly enhanced after a longer administration duration (3 h). The results of CLSM were consistent with the data of accumulated PpIX extracted from the tissues, which showed a more significant fluorescence in emulsions than in the free form. The fluorescence DOI 10.1002/jps
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gradient in the control decreased with tissue thickness from the surface to the upper lamina propria. The use of emulsions as the ALA carrier can push the drug to the deeper layer. The first barrier to drug diffusion in the bladder tissue, following administration in the lumen, is the glycosaminoglycan (GAG) layer.38 The hydrophilic nature of the aqueous droplets of emulsions may have an affinity for GAG, giving the droplets access to the umbrella cell layer. Factors contributing to the increased permeation by the emulsions may be the large surface area, and due to the small droplet size. The availability of the drug can be enhanced because the small size ensures close contact with the urothelium, and hence, improves contact with the bladder surface. Wetting and spreading can be enhanced by the low surface tension of emulsions.39 This study examined the surface tension of emulsions using the Wilhemy plate method (Kyowa CBVP-A3, Saitama, Japan), and a value of 25–30 dyn/cm was obtained (72 dyn/cm for water). Due to the higher ALA concentration gradient on the bladder surface, the drug molecules were transported towards the urothelium from the emulsions. The possible mechanisms for governing ALA permeation by emulsions may include sustained release ability, increase of retention time, increase of drug stability, affinity to GAG layer, and the enhancer effect. A previous study40 indicated a safe use of Tween 80 as a vehicle for cisplatin instillation. Tween 80 can also efficiently enhance cisplatin permeation across the bladder wall, which suggests a possible enhancer effect by the emulsions with Tween 80. Further study is necessary to explore or rule out these mechanisms. One immediate instillation after TUR is recommended in all patients. In low-risk patients, no further treatment is needed before recurrence.41 Hence, one instillation of the emulsions for a 3-h dwell time was performed to examine the preliminary safety to bladder tissue. Some detachment of tissues treated with Brij 98-containing systems (E3) may reduce the barrier function of the urothelium, thus making the urothelium more permeable to ALA. This was confirmed by the more-intense fluorescence signal in the confocal image of the superficial layer by Brij 98-containing emulsions. This disruption is possibly reversible, due to the mild and negligible change. The histological examination of treated tissues revealed no overt signs of toxicity or irritation associated with emulsion application. Sustained-retention delivery platforms can serve as drug depots, thereby extending the drug exposure in the bladder cavity beyond the voiding of urine. For a previous case, Le Visage et al.42 developed paclitaxel-loaded microspheres, which can be retained on the bladder wall for more than 48 h. Similarly, a solution-state thermosensitive copolymer JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010
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is capable of transforming itself into a gel matrix at 378C, showing sustained drug release and retention in bladders over multiple bladder voidings.18 Our preliminary report may support the possibility of intravesical w/o emulsions for prolonging drug retention and increasing delivery depth in bladder. Among four systems examined, the standard formulation (E1) may be most suited for the future development because of its moderate drug delivery, high cytotoxicity, and negligible toxicity on the bladder wall. Further investigation, especially pharmacodynamic evaluation, is of course required to demonstrate the clinical utility of this system.
REFERENCES 1. Witjes JA, Hendricksen K. 2008. Intravesical pharmacotherapy for non-muscle-invasive bladder cancer: A critical analysis of currently available drugs, treatment schedules, and long-term results. Eur Urol 53:45–52. 2. Shen Z, Shen T, Wientjes MG, O’Donnell MA, Au JLS. 2008. Intravesical treatments of bladder cancer: Review. Pharm Res 25:1500–1510. 3. Giannantoni A, Di Stasi SM, Chancellor MB, Costantini E, Porena M. 2006. New frontiers in intravesical therapies and drug delivery. Eur Urol 50:1183–1193. 4. Dalton JT, Yates CR, Yin D, Straughn A, Marcus SL, Golub AL, Meyer MC. 2002. Clinical pharmacokinetics of 5-aminolevulinic acid in healthy volunteers, and patients at high risk for recurrent bladder cancer. J Pharmacol Exp Ther 301:507–512. 5. de Vere White RW. 2006. Transurethral resection of superficial bladder cancer using 5-aminolevulinic acid: Are there any longterm benefits? Nat Clin Pract Urol 3:254–255. 6. Babjuk M, Soukup V, Petrik R, Jirsa M, Dvora´ cek J. 2005. 5aminolaevulinic acid-induced fluorescence cytoscopy during transurethral resection reduces the risk of recurrence in stage Ta/T1 bladder cancer. BJU Int 96:798–802. 7. Denzinger S, Burger M, Walter B, Knuechel R, Roessler W, Wieland WF, Filbeck T. 2007. Clinically relevant reduction in risk of recurrence of superficial bladder cancer using 5-aminolevulinic acid-induced fluorescence diagnosis: 8-year results of prospective randomized study. Urology 69:675–679. 8. Lee SJ, Kim SW, Chung H, Park YT, Choi YW, Cho YH, Yoon MS. 2005. Bioadhesive drug delivery system, using glyceryl monooleate for the intravesical administration of paclitaxel. Chemotherapy 51:311–318. 9. Bunke A, Zerbe O, Schmid H, Burmeister G, Merkle HP, Gander B. 2000. Degradation mechanism and stability of 5aminolevulinic acid. J Pharm Sci 89:1335–1341. 10. Tyagi P, Wu PC, Chancellor M, Yoshimura N, Huang L. 2006. Recent advances in intravesical drug/gene delivery. Mol Pharm 3:369–379. 11. Solans C, Izquierdo P, Nolla J, Azemar N, Garcia-Celma M.J. 2005. Nano-emulsions. Curr Opin Colloid Interface Sci 10:102– 110. 12. Wu H, Ramachandran C, Bielinska AU, Kingzett K, Sun R, Weiner ND, Roessler BJ. 2001. Topical transfection using plasmid DNA in a water-in-oil nanoemulsion. Int J Pharm 221:23–34. 13. Wang JJ, Hung CF, Yeh CH, Fang JY. 2008. The release and analgesic activities of morphine and its ester prodrug, morphine propionate, as formulated by water-in-oil nanoemulsions. J Drug Target 16:294–301. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010
14. Kirchmann H, Pettersson S. 1994. Human urine: Chemical composition and fertilizer use efficiency. Nutr Cycl Agroecosyst 40:149–154. 15. Oishi H, Nomiyama H, Nomiyama K, Tomokuni K. 1996. Fluorometric HPLC determination of 5-aminolevulinic acid in the plasma and urine of lead workers: Biological indicators of lead exposure. J Anal Toxicol 20:106–110. 16. Shen SC, Lee WR, Fang YP, Hu CH, Fang JY. 2006. In vitro percutaneous absorption and in vivo protoporphyrin IX accumulation in skin and tumors after topical 5-aminolevulinic acid application with enhancement using an erbium: YAG laser. J Pharm Sci 95:929–938. 17. Datta SN, Allman R, Loh CS, Mason M, Matthews PN. 1997. Photodynamic therapy of bladder cancer cell lines. Br J Urol 80:421–426. 18. Tyagi P, Li Z, Chancellor M, De Groat WC, Yoshimura N, Huang L. 2004. Sustained intravesical drug delivery using thermosensitive hydrogel. Pharm Res 21:832–837. 19. Madhusudhan B, Rambhau D, Apte SC, Gopinath D. 2007. 1-OAlkylglycerol stabilized carbamazepine intravenous o/w nanoemulsions for drug targeting in mice. J Drug Target 15:154– 161. 20. Hung CF, Chen JK, Liao MH, Lo HM, Fang JY. 2006. Development and evaluation of emulsion-liposome blends for resveratrol delivery. J Nanosci Nanotechnol 6:2950–2958. 21. Yamaguchi T. 1996. Lipid microspheres as drug carriers: A pharmaceutical point of view. Adv Drug Deliv Rev 20:117–130. 22. Buszello K, Harnisch S, Mu¨ ller RH, Mu¨ ller BW. 2000. The influence of alkali fatty acids on the properties and the stability of parenteral o/w emulsions modified with Solutol HS151. Eur J Pharm Biopharm 49:143–149. 23. Spernath A, Aserin A, Ziserman L, Danino D, Garti N. 2007. Phosphatidylcholine embedded microemulsions: Physical properties and improved Caco-2 cell permeability. J Control Release 119:279–290. 24. Bouchemal K, Brianc¸on S, Perrier E, Fessi H. 2004. Nanoemulsion formation using spontaneous emulsification: Solvent, oil and surfactant optimization. Int J Pharm 280:241–251. 25. Wang JJ, Sung KC, Hu OYP, Yeh CH, Fang JY. 2006. Submicron lipid emulsion as a drug delivery system for nalbuphine and its prodrugs. J Control Release 115:140–149. 26. Mason TG. 2000. Estimating the viscoelastic moduli of complex fluids using the generalized Stokes-Einstein equation. Rheol Acta 39:371–378. 27. Nielsen HM, Aemisegger C, Burmeister G, Schuchter U, Gander B. 2004. Effect of oil-in-water emulsions on 5-aminolevulinic acid uptake and metabolism to PpIX in cultured MCF-7 cells. Pharm Res 21:2253–2260. 28. Elfsson B, Wallin I, Eksborg S, Rudaeus K, Ros AM, Ehrsson H. 1998. Stability of 5-aminolevulinic acid in aqueous solution. Eur J Pharm Sci 7:87–91. 29. Bjerregaard S, So¨ derberg I, Vermehren C, Frokjaer S. 1999. Formulation and evaluation of release and swelling mechanism of a water-in-oil emulsion using factorial design. Int J Pharm 193:1–11. 30. Yoshioka T, Florence AT. 1994. Vesicle (noisome)-in-water-inoil (v/w/o) emulsions: An in vitro study. Int J Pharm 108:117– 123. 31. Chung H, Kim TW, Kwon M, Kwon IC, Jeong SY. 2001. Oil components modulate physical characteristics and function of the natural oil emulsions as drug or gene delivery system. J Control Release 71:339–350. 32. Spernath A, Aserin A. 2006. Microemulsions as carriers for drugs and nutraceuticals. Adv Colloid Interface Sci 128– 130:47–64. 33. Jager-Lezer N, Terrisse I, Bruneau F, Tokgoz S, Ferreira L, Clausse D, Seiller M, Grossiord JL. 1997. Influence of lipophilic surfactant on the release kinetics of water-soluble molecules DOI 10.1002/jps
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entrapped in a w/o/w multiple emulsion. J Control Release 45:1–13. Bjerregaard S, So¨ derberg I, Vermehren C, Frokjaer S. 1999. The effect of controlled osmotic stress on release and swelling properties of a water-in-oil emulsion. Int J Pharm 183:17–20. Vasir JK, Labhasetwar VD. 2005. Nanosystems in drug targeting: Opportunities and challenges. Curr Nanosci 1:47–64. Hatefi A, Amsden B. 2002. Camptothecin delivery methods. Pharm Res 19:1389–1399. Lu Z, Yeh TK, Tsai M, Au JLS, Wientjes MG. 2004. Paclitaxelloaded gelatin nanoparticles for intravesical bladder cancer therapy. Clin Cancer Res 10:7677–7684. Grabnar I, Bogataj M, Belicˇ A, Logar V, Karba R, Mrhar A. 2006. Kinetic model of drug distribution in the urinary bladder wall following intravesical instillation. Int J Pharm 322:52–59.
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39. Tadros T, Izquierdo P, Esquena J, Solans C. 2004. Formation and stability of nano-emulsions. Adv Colloid Interface Sci 108– 109:303–318. 40. Wagner HA, Groebe G, Engelmann U. 1988. Serum and tissue levels of platinum after cisplatinum instillation of the rat bladder. Urol Res 16:85–88. 41. Sylvester RJ, Oosterlinck W, Witjes JA. 2008. The schedule and duration of intravesical chemotherapy in patients with nonmuscle-invasive bladder cancer: A systematic review of the published results of randomized clinical trials. Eur Urol 53: 709–719. 42. Le Visage C, Rioux-Leclercq N, Haller M, Breton P, Malavaud B, Leong K. 2004. Efficacy of paclitaxel released from bioadhesive polymer microspheres on model superficial bladder cancer. J Urol 171:1324–1329.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010
Faculty of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan
3
Department of Pathology, College of Medicine, Taipei Medical University, Taipei, Taiwan
Received 31 March 2009; revised 27 September 2009; accepted 29 September 2009 Published online 17 November 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.22006 ABSTRACT: The present work reports on the development of water-in-oil (w/o) emulsions for the intravesical administration of 5-aminolevulinic acid (ALA). The physicochemical properties of droplet size, zeta potential, and viscosity of the emulsions are characterized and the ability of the emulsions to release ALA following in vitro application is tested. The delivery systems are administered intravesically for 1 and 3 h in rats to examine the drug accumulation in bladder tissue. The mean size and zeta potential of the emulsions are 50–200 nm and 3 to 14 mV, respectively. The loading of ALA into the emulsions resulted in a slower and sustained release. The release extent was found to be inversely related to the droplet size of the emulsions. The emulsions did not increase the drug permeation into tissues during short exposure duration (1 h). When the dwell time was extended to 3 h, the systems showed a 2.7-fold increase in the ALA concentration in the bladder wall. Images of confocal laser scanning microscopy demonstrated a higher and deeper fluorescence signal, with emulsion administration, as compared to the aqueous control. Intravesical emulsion delivery provides a significant advantage for drugs targeting bladder tissues. ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:2375–2385, 2010
Keywords:
emulsion; 5-aminolevulinic acid; intravesical delivery; bladder; sustained release
INTRODUCTION Bladder cancer is the fourth most common malignancy among men in Western countries, and the high recurrence rate makes it the most prevalent malignancy.1 A standard treatment for patients with bladder cancer is surgical transurethral resection (TUR) of the tumor. Intravesical chemotherapy is widely used as adjuvant therapy after TUR, to reduce the risk of recurrence.2 The intravesical route permits site-specific delivery of drugs, with a reduced side-effect profile compared to oral delivery systems, either by avoiding first-pass metabolism or by obtaining a local effect.3 Intravesical delivery is an administration method of inserting a catheter from the urinary tract to the bladder cavity to deliver drugs. 5-Aminolevulinic acid (ALA) is a precursor of protoporphyrin IX (PpIX), which is used in photoCorrespondence to: Jia-You Fang (Telephone: þ886-3-2118800 ext 5521; Fax: þ886-3-2118236; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 99, 2375–2385 (2010) ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association
diagnosis and photodynamic therapies (PDT) of malignant bladder cancer.4,5 It combines photosensitizers that selectively bind to tumor tissues and a powerful intravesical light source to destroy the complex formed by the tumor cell and photosensitizer.1 ALA can reduce the recurrence rate in stage Ta/ T1 bladder transitional cell carcinoma.6 It also helps to detect the location of bladder tumor for clinical use.7 ALA is a hydrophilic molecule with a molecular weight of 167.7. The efficacy of intravesical therapy for bladder cancer is in part limited by the poor penetration of hydrophilic drugs into the urothelium.8 The half-life of ALA in the body is quite short (45 min) due to its insufficient stability under physiological conditions.9 Nanosystems with a welldefined particle size and shape have immense potential for intravesical delivery as they can enhance the ability of drugs to cross the urothelium and prolong drug retention near the tumor site.10 Moreover, the encapsulation of drugs into nanosystems can protect the molecules, and thus, prevent instability. Emulsions stabilized with nonionic
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surfactants are a class of nanosystems with a droplet size of 20–200 nm.11 Water-in-oil (w/o) nanoemulsions are considered to be attractive prolonged release systems for hydrophilic drugs, with the oil and interfacial layers acting as a release barrier.12,13 The aim of this study was to develop w/o emulsions as intravesical delivery systems to deliver ALA to the bladder for antitumor therapy. The mean droplet size, surface charges, and viscosity were evaluated to determine the physicochemical characteristics of the emulsions. The drug release properties of the emulsions were determined in vitro using a Franz cell. ALA permeation into the bladder wall after intravesical administration was examined using rats as an animal model. The possible disruption of normal bladder tissue by these w/o emulsions was also investigated for safety reasons.
of the emulsions are shown in Table 1. Under the processing temperature (508C), ALA molecules could remain stable without degradation by immersing a 0.1% ALA in double-distilled water at 508C for 30 min. The amount of ALA in water before and after the heating was quantified by HPLC. Size and Zeta Potential of Emulsions The mean size (z-average) and zeta potential of the emulsions were measured by photon correlation spectroscopy (Malvern Nano ZS1 90, Worcestershire, UK), using a helium–neon laser with a wavelength of 633 nm. The formulations were diluted fivefold with sesame oil before measurement. Photon correlations of spectroscopic measurements were carried out at a scattering angle of 908. The stability of emulsions was determined by monitoring size and zeta potential at 378C and 65% relative humidity for 28 days.
MATERIALS AND METHODS
Viscosity of Emulsions
Materials
The viscosity of the emulsions was determined using a programmable viscometer (DV-IIþ, Brookfield, Middleboro, MA). The spindle (s21, 170 mm) was rotated in the samples, and viscosity was recorded 60 s after rotation commenced, when the level had stabilized. The speed of the paddle was set to 30 rpm.
5-Aminolevulinic acid (ALA), sesame oil, and Span 80 were purchased from Sigma Chemical (St. Louis, MO). Tween 80 was obtained from Kanto Chemical (Tokyo, Japan). Plurol diisostearique (Plurol) was obtained from Gattefosse´ (Gennevilliers, France). Brij 98 was provided by Acros Organics (Geel, Belgium). Hydrogenated soybean phosphatidylcholine (SPC, Phospholipon1 80H) was supplied by the American Lecithin Company (Oxford, CT). Preparation of w/o Emulsions The oil and aqueous phases were separately prepared. The oil phase consisted of oil and surfactants, while the aqueous phase consisted of double-distilled water and the drug (to achieve a concentration of 0.1% (w/v) in the final product). The two phases were heated separately to 508C. The oil phase was further mixed using a high-shear homogenizer (Pro250, Pro Scientific, Monroe, CT) at 12,000 rpm for 10 min. The diameter of the generator was 5 mm. Then, the oil phase was added to the aqueous phase and sonicated using a probe-type sonicator (VCX600, Sonics and Materials, Newtown, CT) for 10 min at 35 W. The total volume of the final product was 10 mL. Compositions
In Vitro ALA Release ALA release from the w/o emulsions was measured using a Franz diffusion cell. A cellulose membrane (CelluSep1 T3, with a molecular weight cutoff of 3500) was mounted between the donor and receptor compartments. The donor medium consisted of 0.5 mL of vehicle containing ALA. The receptor medium (5.5 mL) consisted of pH 6 citrate-phosphate buffer. The available diffusion area between the cells was 0.785 cm2. The stirring rate and temperature were set to 600 rpm and 378C, respectively. At appropriate intervals, 300-mL aliquots of the receptor medium were withdrawn, and then immediately replaced with an equal volume of fresh buffer. The amount of ALA released was determined by highperformance liquid chromatography (HPLC). In the study examining the influence of urine on ALA release, 0.5 mL of synthetic urine was added to the
Table 1. The Compositions and Their Percentages (%, v/v) of Water-in-Oil Emulsions Used in This Study Code E1 E2 E3 E4
Water
Sesame Oil
Span 80
Tween 80
Plurol
Brij 98
SPC
10 10 10 10
44 44 44 44
30 — 30 28
16 16 — 16
— 30 — —
— — 16 —
— — — 2
SPC, soybean phosphatidylcholine. The hydrophile–lipophile balance (HLB) of Span 80, Tween 80, Plurol, and Brij 98 is 4.3, 15.0, 4.5, and 15.3, respectively. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010
DOI 10.1002/jps
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donor compartment. The synthetic urine solution contained 14.1 g NaCl, 2.8 g KCl, 17.3 g urea, 1.9 mL of a 25% (v/v) ammonia solution, 0.6 g CaCl2, and 0.43 g of MgSO4 made up to 1 L with water.14 HPLC Analysis of ALA The fluorescence derivation of ALA samples was based on a modification of the Hantzsch reaction.15 The ALA contents of samples were analyzed by HPLC, as cited previously.16 Cytotoxicity Assay
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PpIX in the bladder tissue was extracted by a homogenization method. One milliliter of a 1.5 M perchloric acid/methanol (1:1, v/v) mixture was added to the excised tissue in a glass homogenizer and ground for 5 min. The resulting solution was centrifuged for 10 min at 10000 rpm. The fluorescence of the supernatant was determined spectrofluorometrically (Hitachi F-2500, Tokyo, Japan) at an excitation wavelength of 403 nm and emission at 602 nm. Confocal Laser Scanning Microscopy (CLSM) Bladder samples obtained following in vivo instillation were examined for PpIX fluorescence images by CLSM. The thickness of the bladder wall was optically scanned at 10-mm increments through the Z-axis of a Leica TCS SP2 confocal microscope (Wetzlar, Germany). Optical excitation was carried out with a 543-nm He–Ne laser beam, and fluorescence emission was detected at 560–620 nm.
Human urothelial carcinoma cell line (T24) was purchased from the American Type Culture Collection (Rockville, MD). The cells were seeded at an initial concentration of 1 105 cells/well in a 24-well plate, and incubated in a 1 mL medium (10% fetal bovine serum, 89% RPMI 1640, and 1% penicillin– streptomycin). Water and emulsions, with or without ALA diluted with medium, was added at 24 h postinoculation, and plates were incubated in a 5% CO2 atmosphere at 378C. After 4 h of incubation, each plate was then exposed to light from a tungstenhalogen lamp (Osram Decostar51, 50 W) for 40 min. The total spectral irradiance at the level of the cells was 10 mW/cm2, as measured with a light meter (illumination meter). Subsequently the medium was continuously incubated for 72 h. After PBS washing, cells were incubated with 5 mg/mL 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) in RPMI 1640 for 2 h at 378C. Formozan crystals resulting from MTT reduction were dissolved by adding 200 mL DMSO and gently agitated for 30 min. The absorbance of supernatant was then measured spectrophotometically in an ELISA reader (Fluostar Optima, BMG Labtech GmbH, Offenburg, Germany) at 550 nm. Cell viability was calculated as the percentage of the control.
RESULTS
In Vivo Intravesical Instillation into Bladder Tissues
Physicochemical Characterization of the Emulsions
Female Sprague–Dawley rats, weighing 225–250 g, were used in this study. The animal protocol was approved by the Institutional Animal Care and Use Committee of Chang Gung University. Animals were anesthetized with urethane (1.2 g/kg) subcutaneously before intravesical instillation. The residual urine was evacuated by pressing the lower abdomen. A polyurethane catheter (BD Angiocath Plus1, Becton Dickinson Korea, Gyeongbuk, Korea) was inserted into the bladder. The bladder was washed with 0.5 mL normal saline. Subsequently 0.2 mL of emulsions was instilled into the bladder. The catheter was removed and the urethra was ligated with cotton thread. The dwell time for instillation was 1 and 3 h. After the application time, the bladder was excised and washed with saline.
The droplet size of the w/o emulsions ranged 50–200 nm, as shown in Table 2, which fall in the range of so-called nanoemulsions (20–200 nm). For the strict definition of nanotechnology, a size of <100 nm is feasible for qualification as a nanosystem. Hence the final products prepared in this work are nano- or submicron-sized emulsions. There was a difference in the average size of the droplets, determined according to the types of emulsifier used. The standard formulation (E1) was stabilized by a combination of two surfactants: a sorbitan mono-9octadecenoate (Span 80) and a polyoxyethylene 20 sorbitan monooleate (Tween 80). The size analysis indicated that this nanoemulsion had a mean droplet size of 108 nm. Span 80 with a hydrophile–lipophile balance (HLB) of 4.3 was replaced with Plurol
DOI 10.1002/jps
Histological Examination of Bladder Tissues Immediately after treatment with an aqueous solution, or the w/o emulsions by intravesical instillation for 3 h, a specimen of the exposed area was taken for histological examination. Each specimen was fixed in a 10% buffered formaldehyde solution (pH 7.4) for at least 48 h, and stained with hematoxylin and eosin. For each sample, three different sites were examined and evaluated under light microscopy (Eclipse 4000, Nikon, Tokyo, Japan). Statistical Analysis Statistical analysis of differences between different treatments was performed using unpaired Student’s t-test, and a 0.05 level of probability was taken as the level of significance.
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Table 2. The Characterization of Water-In-Oil Nanoemulsions by Droplet Size and Zeta Potential
Code
Size (nm) at Day 0
Size (nm) at Day 28
Zeta Potential (mV) at Day 0
Zeta Potential (mV) at Day 28
Viscosity (mP s)
pH
E1 E2 E3 E4
108.3 10.7 194.3 18.2 50.4 2.9 209.9 9.6
86.6 8.6 176.6 3.7 38.0 0.5 170.5 10.0
7.4 0.6 10.0 0.7 2.5 0.2 13.6 0.9
10.3 2.6 8.7 0.6 1.6 0.7 10.2 2.4
105.2 5.0 204.2 1.8 188.7 11.9 262.7 11.1
5.2 7.0 4.9 6.0
Each value represents the mean SD (n ¼ 3).
(polyglyceryl diisostearate), with an HLB of 4.5 in the emulsions (E2). This substitution led to an increase in size to 194 nm ( p < 0.05). Brij 98, another hydrophilic emulsifier with an HLB of 15.3, was substituted for Tween 80 (with an HLB of 15.0) to produce an emulsion (E3) with a reduced size of 50 nm ( p < 0.05). SPC was also incorporated into the w/o interface of emulsions as a coemulsifier (E4). Results indicated that a significant enlargement in size (210 nm) occurred after SPC incorporation (E1 vs. E4, p < 0.05). As depicted in Table 2, the zeta potentials of the emulsions ranged from 2.5 to 13.6 mV. SPCcontaining emulsions (E4), which showed the highest zeta potential, as compared to other systems ( p < 0.05). The addition of Plurol resulted in a slight but significant increase (E1 vs. E2, p < 0.05) in surface charge. On the other hand, Brij 98 shielded the zeta potential (E1 vs. E3, p < 0.05). The local retention condition of tissue is an important parameter for drug delivery by emulsions (11). The viscosity/rheological properties are factors affecting retention ability. As summarized in Table 2, the value of viscosity depends on the ingredients used in the emulsion formulations. The respective replacement of Span 80 and Tween 80 (E1) by Plurol (E2) and Brij 98 (E3) increased the viscosity 2-fold. Adding SPC to the systems (E4) further increased the viscosity 2.5fold. This may indicate that Plurol, Brij 98, and SPC themselves possessed a greater viscosity as compared to Span 80 and Tween 80. After 28 days of storage at 378C, the mean diameter of E1 and E2 was maintained (Tab. 2). The presence of Brij 98 and SPC (E3 and E4) slightly, but significantly, decreased ( p < 0.05) the size after storage. Concerning the zeta potential, there were no or negligible changes observed for all formulations from storage.
and continuous release. The release of ALA from the emulsions followed the Higuchi diffusion controlled model equation (release percentage: h1/2). The result showed that the ingredients are important for modulating ALA release. The substitution of Span 80 (E1) by Plurol (E2) further slowed the ALA release. The same phenomenon was observed by incorporating SPC (E4) into the systems. Only 6% of the drug dose had been released from the donor compartment after a 12-h application. Emulsion systems, as prepared with Span 80 and Brij 98 (E3), produced a 2.6-fold enhancement in the
In Vitro ALA Release The in vitro release behavior of ALA is shown in Figure 1A. Release of ALA from double-distilled water was studied as a control group. As can clearly be seen in Figure 1A, ALA release from the water was rapid, with 60% released after 4 h, whereas, the release behavior from the w/o emulsions exhibited a slower JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010
Figure 1. In vitro release percentage (%) of 5-aminolevulinic acid across a cellulose membrane from double-distilled water (control) and emulsion systems (A) and in the presence of synthetic urine (0.5 mL) in the donor compartment (B). All data are presented as the mean SD of four experiments. DOI 10.1002/jps
DELIVERY OF ALA FROM W/O NANO/SUBMICRON-EMULSION SYSTEMS
released amount of ALA, with respect to the standard emulsion (E1). Synthetic urine was added to the donor phase and mixed with the emulsions in order to simulate in vivo conditions of urine infused into the bladder. ALA release in the presence of urine is demonstrated in Figure 1B. The percentage of released ALA from the aqueous solution (control) was reduced in the presence of synthetic urine, possibly due to the dilution effect. The standard emulsion (E1), and the systems with Brij 98 (E3) also showed decreased ALA release after loading with urine. This reduction was negligible in the formulations with Plurol and SPC (E2 and E4, p > 0.05). Cytotoxicity Assay To assess the cytotoxicity of ALA-loaded emulsions, their tumor-killing activity was determined against an urothelium cancer cell lining by MTT assay. The effect of light exposure on the viability of cells by free ALA solution was first examined, as presented in Figure 2A. The PDT assembly was set according to
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the previous study.17 Cells exposed to ALA but no light showed a limited decrease in cell death. The results of survival after PDT of the cells exhibited that the light is essential to induce proliferation inhibition. All further experiments were performed under PDT treatment. As shown in Figure 2B, the emulsions with Brij 98 (E3) completely inhibited the growth of urothelial carcinoma cells in both doses (0.3 and 0.5 mM). The standard system (E1) also showed a significant cytotoxicity to the cells. Antiproliferative activity was negligible ( p > 0.05) for the vehicle with Plurol (E2) at a dose of 0.3 mM. The incorporation of SPC in the system (E4) led to the attenuation of the antiproliferative activity of the emulsions. Generally, the cytotoxicity increased in a trend of E3 > E1 > E4 > E2. Empty nanosystems, without ALA, were also tested, and it was found that the vehicles themselves had no effect on cytotoxicity. This indicates that the cytotoxicity toward the urothelial carcinoma cells was mainly a consequence of the ALA molecules. In Vivo Intravesical Instillation into Bladder Tissues Rats received an intravesical dose of ALA to examine the drug amounts in the bladder wall after 1- and 3-h applications. The emulsions with Plurol (E2) were not included in this experiment because of the extremely low ALA release and cytotoxicity from this emulsion. ALA is converted to PpIX after permeation into the bladder wall. As shown in Figure 3, PpIX accumulation from the aqueous control was 1.22 and 0.54 nmol/g bladder tissue with 1- and 3-h dwell times, respectively. The accumulation at 1 h from w/o
Figure 2. Viability (%) of human urothelial carcinoma cell line (T24) following treatment with free ALA in water, with or without light exposure (A), and emulsion systems with light. (B) Each value represents the mean SD of five experiments. DOI 10.1002/jps
Figure 3. In vivo protoporphyrin IX (PpIX) accumulation (nmol/g) in bladder tissues after intravesical instillation of double-distilled water (control) and emulsions with 5-aminolevulinic acid (0.1%) for 1 and 3 h. All data are presented as the mean SD of six experiments. Significantly higher ( p < 0.05) as compared to the control at the same time plot; Significantly lower ( p < 0.05) as compared to the control at the same time plot. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010
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emulsions was only one-half of the accumulation from the control. There were no significant differences ( p > 0.05) among the accumulation levels of the three emulsions at 1 h. The PpIX concentration at 3 h showed a contrary result, as compared to that at 1 h. The standard emulsions (E1) produced a 2.7-fold enhancement in the average concentration of total PpIX in tissue sections. Changes in the hydrophilic emulsifier from Tween 80 to Brij 98 (E3) did not significantly change ( p > 0.05) the PpIX accumulation at 3 h. On the other hand, PpIX permeation decreased slightly, but significantly ( p < 0.05) when SPC was added to the formulations (E4). CLSM was utilized to observe the superficial PpIX distribution in the bladder wall. The superficial layer was optically scanned at 10-mm increments for nine fragments from the surface of the mucosa, as shown in Figure 4 (left to right, top to bottom). The total 90 mm comprises the urothelium and a part of the lamina propria. Figure 4 shows confocal images, as obtained from the controls and w/o emulsions after ALA instillation for 3 h. An increase in red fluorescence at 488-nm excitation and 590-nm emission was observed and considered to be PpIX accumulation. Compared to the control (Fig. 4A), the increase in red fluorescence in the superficial layer, as treated with emulsions (Fig. 4B and C), can clearly be seen with intense signals. Only a pale signal was detected in the first three images (30 mm) of the control group. The ALA loaded in the emulsions could have permeated the deeper layers. The emulsions with Brij 98 (E3, Fig. 4C) produced an even stronger signal than the standard formulations (E1, Fig. 4B), although there were no significant differences ( p > 0.05) between their PpIX accumulation levels extracted from bladder tissue. Histological Examination of Bladder Tissues A preliminary safety profile of emulsions on bladder tissue was evaluated by checking the histology, as depicted in Figure 5. Light microscopy indicated no observable damage to the whole bladder wall in the control group (Fig. 5A). It can be seen that the bladder wall is divided into three sections: the urothelium, which is not blood-perfused; the underlying lamina propria; and muscle layer of the bladder, which contains blood vessels and lymphatics. Photographs taken after staining the bladder treated with standard nanoemulsions (E1) revealed no adverse histological changes (Fig. 5B). The urothelium remained intact after a 3-h treatment with this system. When the bladder was treated with the instilled emulsions containing Brij 98 (E3), there was generally no significant change in the structures (Fig. 5C). However, it is noted that there was some JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010
Figure 4. Confocal laser scanning microscopic (CLSM) micrographs of rat bladder tissues after the in vivo intravesical instillation of 5-aminolevulinic acid (0.1%) for 3 h from (A) double-distilled water (control), (B) standard emulsions (E1), and (C) emulsions with Brij 98 (E3) (original magnification, 20). The specimen was viewed by CLSM at 10-mm increments through the Z-axis from the surface to the deeper layers (top to bottom).
detached superficial urothelium (Fig. 5D, depicted by green arrows). There was also slight degeneration of the urothelium (depicted by blue arrows). These changes were an early and mild disruption of the bladder structure. It can be concluded that the emulsions with Tween 80 (E1) showed a safer delivery to the bladder, as compared to that of Brij 98 (E3). DOI 10.1002/jps
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Figure 5. Histological examination of rat bladder tissues after in vivo intravesical instillation for 3 h with (A) double ¼ distilled water (control), (B) standard emulsions (E1), (C) emulsions with Brij 98 (E3), and (D) emulsions with Brij 98 (E3) in another view (original magnification, 200).
DISCUSSION Intravesical drug delivery continues to remain an ideal treatment option for therapy of superficial bladder cancer. However, conventional vehicles fail to provide sustained exposure or enhanced permeation of drugs to the urothelium.18 Emulsions have been widely explored as potential drug delivery systems. In the present work, nano- or submicronsized emulsions with an optimal formulation design provided controlled, sustained release of ALA. It was found that ALA permeation into the urothelium was enhanced by encapsulation into the emulsions, especially in the presence of urine in the bladder. The drug delivery potential of emulsions depends on the physicochemical properties of the droplets, such as the size, surface charge, and viscosity.19 Incorporation of Plurol (E2), instead of Span 80 (E1), led to significant increase in the droplet size. This may have been due to the spacious hydrophobic chains of Plurol, which have two stearic acid chains. The hydrophilic polyglycerol of Plurol was bound into the interface, whereas the fatty acid chains remained at the surface of the droplets. The droplet size can be increased following an increase in the interfacial film thickness. A similar phenomenon is shown in systems with SPC (E4), the structure of which possesses two alkyl chains. Brij 98 may have the ability to strengthen droplet integrity,20 resulting in the strong DOI 10.1002/jps
cohesion of the droplet and the avoidance of aggregation. Another possibility is the influence of HLB. The emulsifier system with a determined HLB may well have stabilized the emulsions which had a moderate polarity. The mixture of Span 80 and Brij 98 may facilitate a satisfactory emulsification for the whole system. Brij 98-containing systems (E3) were the only emulsion that was actually in the nano-scale (<100 nm). The physical stability of a nanosystem is one of the most important desired product characteristics. Emulsions developed in this study generally showed acceptable storage stability, after 28-day duration, although a slight decrease of the size was observed for E3 and E4. The aggregation or fusion of the droplets was always detected after storage, however, the size reduction was also possible from the destruction of micro- or nanoparticles, which is not uncommon during storage.21 Another possibility is the free fatty acids derived from sesame oil during storage. Fatty acids with some surfactant characteristics might reside in the w/o interfacial membranes, strengthening the emulsifying ability of the whole system, thus, reducing the droplet size. Further experimentation is necessary to confirm this hypothesis. Both Span 80 and Tween 80 are nonionic species. The negative charge in the interface is believed to be the result of the ionization of the free fatty acids derived from the hydrolysis of triglycerides in the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010
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sesame oil. Alkali fatty acids are thought to yield mixed films with a higher packing density of interfacial film-forming components.22 SPC in the interface of the emulsions (E4) was responsible for the higher negative surface charges. The major component of SPC (Phospholipon1 80H), as used in this study, was 80% phosphatidylcholine, which exhibits a net charge. The other components were phosphatidylserine, phosphatidic acid, phosphatidylglycerol, and phosphatidylinositol, all of which are negatively charged. These anionic fractions were responsible for the negative zeta potential of SPC-containing emulsions. It can be seen that the system with Plurol (E2) showed a higher negative charge as compared to that with Span 80 (E1). The structure of Plurol belongs to glycerides. The free stearic acids derived from Plurol may contribute to the negative charges within the interface. Further investigation is needed to elucidate this inference. The inner phase with smaller size possesses limited surface area for the droplets. This leads to the lower negative charge of the emulsions. This can explain the lowest zeta potential of the systems containing Brij 98 (E3) because of their smallest size. The viscous and elastic properties of the dispersions are important for their application in the bladder since they may provide a bio-adhesion to the bladder wall, thus prolonging the retention duration. The emulsions with SPC (E4) showed the highest viscosity. Moreover, both E2 and E4, with the larger droplet sizes, exhibited more-viscous properties, as compared to E1 and E3. This may have been due to the hydrophobic hydrocarbon tails of the SPC dangling out of the interface, which resulted in significant entanglements and enhanced viscosity.23 It is well established by experimental results that increasing the viscosity of the emulsion phase leads to an increase in the particle size.24,25 According to the generalized Stokes–Einstein equation developed by Mason,26 the hydrodynamic diameter of a droplet is inversely related to its viscosity. The contradiction may cause only a moderate correlation (Pearson’s correlation coefficient r ¼ 0.81, p ¼ 0.093) to be detected between viscosity and size. This suggests that, not only the size but also other factors, such as the emulsion structure and droplet-to-droplet interactions can influence nanoemulsion viscosity/rheology. For example, a higher number of smaller droplets results in more droplet-to-droplet interactions, and an increased resistance to flow. The main clinical limitation with ALA is its instability in its physiologic pH. ALA is relatively stable at lower pH values.7,27 The shelf-life of ALA, in a pH 7.53 aqueous solution, can be as short as 10 min.28 The inclusion of ALA in the inner phase of the emulsions may protect the molecules from degradation in tissues and urine. The acidic pH of the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010
emulsions, as shown in Table 2, supports good stability of ALA in these systems (E1, E3, and E4). This effect may produce prolonged drug exposure in the urothelium. In addition, the acidic pH increases the ion permeability of the urothelium.3 The release rate generally showed a trend of E3 > E1 > E4 > E2. The release kinetics of the Higuchi model assume that when one layer of w/o emulsion droplets is depleted of drug, release from the next layer begins.13,29 The drug is stably retained in the inner phase for a determined duration, followed by its slow release into the external phase. The oil phase may constitute an additional barrier, which prevented the partitioning of ALA into the continuous phase, thereby limiting the delivery. It is recognized that increasing the viscosity of w/o emulsions causes a more-rigid structure and decreases the rates of drug release.12,30 However, this did not occur in the present study since a low correlation (r ¼ 0.25, p ¼ 0.754) was calculated between the viscosity and released percentage at 12 h. The viscous properties of emulsions may increase the residence time at the mucosal membrane.18 According to the results of in vivo delivery, it seems that viscosity of emulsions is a negligible factor influencing ALA release and permeation. Another possible mechanism predominating ALA delivery is the influence of droplet size. The rate of release generally increases with smaller droplets, since a small-droplet system has a larger total surface area where drug diffusion can occur. Our profiles showed a good correlation (r ¼ 0.92, p ¼ 0.077) between the emulsion size and the released percentage. There is evident that the drug is more easily released with a small droplet size.25,31,32 Significantly reduced drugs released from the emulsions with Plurol (E2) and SPC (E3) were observed. The addition of fatty acid or ester with long alkyl chains, such as sodium stearate, is thought to yield a higher packing in the interfacial film.22,25 Both Plurol and SPC have additional alkyl chains, as compared to Span 80. It was apparent that urine reduced ALA release for most vehicles. A previous study10 suggested that intravesical transport can be adversely affected by dilution of the instilled drug solution by urine in the bladder. Responses to intravesical delivery are directly proportional to the drug concentration rather than the drug dose.18 Hence, the dilution by urine led to a reduction in the concentration of ALA and subsequent drug release. Nanoemulsions with smaller droplets (E1 and E3) showed a greater reduction in ALA release, as compared to the formulations with larger sizes (E2 and E4). It is possible that a water-inoil-in-water (w/o/w) multiple emulsion type was formed by mixing the w/o emulsions with water (urine). In w/o emulsions, the oil phase may be regarded as a semipermeable membrane allowing the DOI 10.1002/jps
DELIVERY OF ALA FROM W/O NANO/SUBMICRON-EMULSION SYSTEMS
transport of water, and this flux of water into the inner phase of the emulsions may then be induced by an osmotic gradient.29 The release of the drug from the droplets is reduced when water enters the aqueous phase and produces an osmotic (swelling) effect. This osmotic downregulation of release has been reported in the case of w/o/w systems.29,33 The swelling downregulation is explained by a reverse solvent drag or by a reduced drug concentration gradient after the swelling of the inner phase. The emulsion with small droplets is more responsive to osmotic pressure than are larger droplets.29,34 The results of the present study are consistent with this theory. The cytotoxicity results showed that free ALA in water induced growth inhibition in the cells and ALA was slowly released from the emulsion droplets. A higher release rate may render a high level of free ALA for cell uptake. It is noticeable that E3 showed even higher antiproliferative activity than the free form ( p < 0.05) at a lower dose (0.3 mM). Hence, the activity against cancer cells might not have completely been due to the direct penetration of free ALA into the cytoplasm. Nanoparticles need to enter cells and diffuse through the viscous cytosol in order to access the particular cytoplasmic targets where the sites of action are located.35 Cellular uptake of the drug can possibly 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.36 Detailed investigation of the emulsion effect on cytotoxicity is not the main focus of this study. The basic mechanism is to be elucidated in future studies. ALA in aqueous solution (control) produced a higher PpIX accumulation ( p < 0.05) in the bladder wall after a 1-h administration due to the burst release of ALA, as confirmed by the in vitro release experiments. However, the accumulation was greatly diminished when the application duration was prolonged to 3 h because of the depletion of the drug as it was released. The duration of drug instillation during intravesical bladder therapy is typically limited to 2 h, after which time urine production is largely responsible for decreasing the drug concentration in the bladder.4,37 The bladder wall is composed of an unperfused transitional epithelium called the urothelium, which represents the toughest known barrier to drug delivery.10,38 Although the emulsions could not improve ALA permeation at the initial stage of application (1 h) because of the slow release from the inner phase, PpIX accumulation was significantly enhanced after a longer administration duration (3 h). The results of CLSM were consistent with the data of accumulated PpIX extracted from the tissues, which showed a more significant fluorescence in emulsions than in the free form. The fluorescence DOI 10.1002/jps
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gradient in the control decreased with tissue thickness from the surface to the upper lamina propria. The use of emulsions as the ALA carrier can push the drug to the deeper layer. The first barrier to drug diffusion in the bladder tissue, following administration in the lumen, is the glycosaminoglycan (GAG) layer.38 The hydrophilic nature of the aqueous droplets of emulsions may have an affinity for GAG, giving the droplets access to the umbrella cell layer. Factors contributing to the increased permeation by the emulsions may be the large surface area, and due to the small droplet size. The availability of the drug can be enhanced because the small size ensures close contact with the urothelium, and hence, improves contact with the bladder surface. Wetting and spreading can be enhanced by the low surface tension of emulsions.39 This study examined the surface tension of emulsions using the Wilhemy plate method (Kyowa CBVP-A3, Saitama, Japan), and a value of 25–30 dyn/cm was obtained (72 dyn/cm for water). Due to the higher ALA concentration gradient on the bladder surface, the drug molecules were transported towards the urothelium from the emulsions. The possible mechanisms for governing ALA permeation by emulsions may include sustained release ability, increase of retention time, increase of drug stability, affinity to GAG layer, and the enhancer effect. A previous study40 indicated a safe use of Tween 80 as a vehicle for cisplatin instillation. Tween 80 can also efficiently enhance cisplatin permeation across the bladder wall, which suggests a possible enhancer effect by the emulsions with Tween 80. Further study is necessary to explore or rule out these mechanisms. One immediate instillation after TUR is recommended in all patients. In low-risk patients, no further treatment is needed before recurrence.41 Hence, one instillation of the emulsions for a 3-h dwell time was performed to examine the preliminary safety to bladder tissue. Some detachment of tissues treated with Brij 98-containing systems (E3) may reduce the barrier function of the urothelium, thus making the urothelium more permeable to ALA. This was confirmed by the more-intense fluorescence signal in the confocal image of the superficial layer by Brij 98-containing emulsions. This disruption is possibly reversible, due to the mild and negligible change. The histological examination of treated tissues revealed no overt signs of toxicity or irritation associated with emulsion application. Sustained-retention delivery platforms can serve as drug depots, thereby extending the drug exposure in the bladder cavity beyond the voiding of urine. For a previous case, Le Visage et al.42 developed paclitaxel-loaded microspheres, which can be retained on the bladder wall for more than 48 h. Similarly, a solution-state thermosensitive copolymer JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010
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is capable of transforming itself into a gel matrix at 378C, showing sustained drug release and retention in bladders over multiple bladder voidings.18 Our preliminary report may support the possibility of intravesical w/o emulsions for prolonging drug retention and increasing delivery depth in bladder. Among four systems examined, the standard formulation (E1) may be most suited for the future development because of its moderate drug delivery, high cytotoxicity, and negligible toxicity on the bladder wall. Further investigation, especially pharmacodynamic evaluation, is of course required to demonstrate the clinical utility of this system.
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