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Design and evaluation of a liposomal delivery system targeting the posterior segment of the eye
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Journal of Controlled Release j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j c o n r e l
Design and evaluation of a liposomal delivery system targeting the posterior segment of the eye Kohei Hironaka a, Yuta Inokuchi b, Yuichi Tozuka a, Masamitsu Shimazawa b, Hideaki Hara b, Hirofumi Takeuchi a,⁎ a b
Laboratory of Pharmaceutical Engineering, Gifu Pharmaceutical University, Japan Laboratory of Biofunctional Evaluation, Molecular Pharmacology, Gifu Pharmaceutical University, Japan
a r t i c l e
i n f o
Article history: Received 17 October 2008 Accepted 17 February 2009 Available online 9 March 2009 Keywords: Liposomes Posterior segment of the eye Retina Atomic force microscopy Eyedrops
a b s t r a c t The purpose of this study was to evaluate the potential of submicron-sized liposomes (ssLips) as a novel system for delivering ocular drugs to the eye's posterior segment. Fluorescence emission of coumarin-6 formulated into ssLip was obvious in that segment in mice after eyedrop administration of the liposomal suspension. Such fluorescence was not observed after administration of either multilamellar vesicles or dimethyl sulfoxide (DMSO) solution containing the same amount of coumarin-6. The highest fluorescence of ssLip occurred 30 min after eyedrop administration, and all fluorescence disappeared after 180 min. The ssLip based on L-α-distearoyl phosphatidylcholine (DSPC ssLip) showed higher fluorescence emission in the retina than that based on egg phosphatidylcholine (EPC ssLip). These results confirmed that the magnitude of fluorescence in the retina was closely related to both liposome rigidity and particle size. Images of the entire eye showed that ssLip was delivered via the non-corneal pathway after administration. The liposomes tested in ocular cells showed little cytotoxicity. These results suggest that ssLip can be used to deliver drugs to the posterior segment of the eye. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The leading causes of vision impairment and blindness are posterior segment-related diseases including age-related macular degeneration, diabetic macular edema and endophthalmitis. Recently, pharmaceutical approaches to these diseases have used steroids and oligonucleotides [1,2]. These drugs are generally administered via invasive methods, such as intravitreal injections and subtenon injections, because noninvasive delivery of drugs is not available. However, repeated injections are associated with potential risks of complications, such as cataracts, vitreous hemorrhages and retinal detachment [3]. Moreover, patients may not comply with such regimens. Thus, there is a pressing need for noninvasive delivery systems targeting the posterior segment of the eye. Systemic administration is one possible way to obviate intravitreal or subtenon injection. One of the disadvantages of systemic administration to deliver drugs to the retina is its efficiency. A very small fraction of the systemically administered dose reaches the ocular tissues because the
⁎ Corresponding author. Gifu Pharmaceutical University, 5-6-1 Mitahora-higashi Gifu 502-8585, Japan. Tel: +81 58 237 8574; fax: +81 68 237 6524. E-mail address: [email protected] (H. Takeuchi). 0168-3659/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2009.02.020
blood-retinal barrier can prevent the drug from entering the retina [4]. Moreover, the doses that are needed in order to have a therapeutic effect via this route can lead to considerable side effects [5]. Topical administration with eyedrops is an alternative way to minimize side effects. However, corneal and conjunctival epithelia, along with tear film, serve as biological barriers to protect the eye from potentially harmful substances and drugs. Therefore, conventional eyedrop formulations usually cannot effectively overcome these barriers. The use of colloidal drug delivery systems, such as nanoparticles, nanoemulsions and liposomes, has received much attention as a way to enhance the bioavailability of drugs administered both systemically and topically. We have recently demonstrated that submicron-sized liposomes (ssLips) penetrated mucosal cells in rat intestine [6]. This finding encouraged us to try to use ssLip in an eyedrop formulation in this study. In ophthalmic therapy, many researchers have investigated the use of liposomes extensively. Liposomes reportedly can come into intimate contact with the ocular surfaces, thus working as barriers, and they can be used to protect drug molecules from metabolic enzymes present at the tear/corneal epithelium interface [7]. Hathout et al. reported that multilamellar liposomes containing acetazolamide were more efficient than acetazolamide solution in lowering intraocular pressure [8]. Shen and Tu reported that the ocular bioavailability of liposomal ganciclovir in liposomes in rabbits was 1.7-fold higher than that of a
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ganciclovir solution [9]. However, there have been no reports on drug delivery to the retina via eyedrops formulating a colloidal drug carrier such as liposomes. The purpose of this study was to evaluate the potential of ssLip as a novel system for delivering ocular drugs to the posterior segment of the eye, including the retina. The behavior of liposomes labeled with coumarin-6 as a fluorescence reagent was investigated after it was topically administered to mice via eyedrops. The cytotoxicity of the liposomes was also tested in vitro using ocular cells. 2. Materials and methods 2.1. Materials Egg phosphatidylcholine (EPC) and L-α-distearoyl phosphatidylcholine (DSPC) were purchased from Nippon Oil and Fats Co., Ltd. (Tokyo, Japan). Dicetyl phosphate (DCP) and cholesterol were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Coumarin-6 as a lipid marker and benzalkonium chloride were purchased from MP Biomedicals LLC (Illkirch, France). 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) was purchased from Nakalai Tesque (Kyoto, Japan). Hank's balanced salt solution (HBSS) was purchased from GIBCO BRL (Grand Island, NY, USA). Mica was purchased from Veeco Co., Ltd. (Tokyo, Japan). All other chemicals were commercial products of reagent grade. 2.2. Preparation of liposomes Multilamellar vesicles (MLV), composed of phospholipid (EPC or DSPC), DCP, and cholesterol at a molar ratio of 8:2:1, were prepared using the hydration method. The lipid mixture containing coumarin-6 was dissolved in a small amount of chloroform in a round-bottom flask and dried in a rotary evaporator under reduced pressure at 40 °C to form a thin lipid film. The film was dried in a vacuum oven overnight to ensure complete removal of the solvent. After addition of HBSS–Hepes buffer, lipid film was incubated in water bath at 70 °C for 30 s, and then vortexed for about 30 s. This cycle was repeated 5 times. The obtained MLV were incubated at 10 °C for 30 min. The ssLip was prepared using an extruder (LipoFast™-Pneumatic; Avestin, Inc., Ottawa, Canada) with a size-controlled polycarbonate membrane (0.1 µm membrane filter pore size; Whatman Japan KK, Tokyo, Japan). Extrusion was performed 41 times under nitrogen pressure (200 psi). The final phospholipid and coumarin-6 concentrations in the resultant liposomal suspension were 20.4 µmol/ml and 0.143 µmol/ ml, respectively. The coumarin-6 entrapment efficiency into ssLip was calculated as follows. The ssLip was obtained by ultrasonication of the MLV followed by filtering the ssLip through a cellulose acetate filter (0.8 µm). The filtered suspensions (0.5 ml) were dissolved in chloroform/methanol (1/1, 3 ml), and the coumarin concentration was measured with fluorometry (F3010, Hitachi, Tokyo, Japan) at an excitation wavelength of 490 nm and an emission wavelength of 520 nm. The entrapment efficiency percentage of ssLip was calculated by the following equation: % entrapment efficiency = Aflt / Aint × 100, where Aflt is the filtered amount of coumarin-6 in ssLip and Aint is the initial amount of coumarin-6 in ssLip. The ssLip particle size was measured with an aliquot of the particulate suspension diluted with a large amount of distilled water by the dynamic light scattering (DLS) method (Zetasizer, Malvern, Worcestershire, UK). The MLV particle size was measured by a laser diffraction size analyzer (LDSA-2400A; Tonichi Computer applications, Tokyo, Japan). The zeta potential of liposomes was measured using a laser Doppler method (Zetasizer, Malvern). Each batch was analyzed in triplicate. Both EPC and DSPC confirmed that more than 98% of coumarin-6 was entrapped into ssLip. As shown in Table 1, there were no differences in particle size, zeta potential or entrapment efficiency of coumarin-6 between the two types of phospholipids.
Table 1 Characterization of liposomes.
EPC MLV DSPC MLV EPC ssLip DSPC ssLip
Mean particle size (nm)
Zeta potential (mV)
5750 6450 125.3 105.4
− 97.5 − 101.1 − 62.9 − 66.2
2.3. Animal studies Un-anesthetized male adult ddY mice (Japan SLC, Hamamatsu, Japan) weighing 30–35 g were used. The mice were fed a regular diet. All experiments were approved and monitored by the Institutional Animal Care and Use Committee of Gifu Pharmaceutical University. A single dose of 3 µl of the liposomal formulation was dropped onto the surface of the left eye. The contralateral eye was used as the control and received no treatment. The mice were then sacrificed 5, 10, 30, 60 or 180 min after administration of the liposomal formulation. Both eyes were enucleated immediately and washed with excess amount of saline, and then fixed overnight in 4% paraformaldehyde at 4 °C. Fixed eyes were immersed in 20% sucrose for 24 h at 4 °C and embedded in optimal cutting temperature compound (Sakura Finetechnical Co., Ltd., Tokyo, Japan). The samples were then sliced with a cryostat (CM1850, Leica Instrument GmbH, Nussloch, Germany) into sections 10 µm thick and placed onto slides under a cover slip. The retinal images, taken at distances between 375 and 625 µm from the optic disc of frozen sections, were observed using epifluorescence microscopy (BX50; Olympus, Tokyo, Japan) with an attached CCD camera (DP30VW, Olympus) and fluorescence filters for coumarin-6 (U-WNIBA, Olympus). In the inner plexiform layer (IPL) at a distance between 475 and 525 µm (50 µm × 50 µm) from the optic disc, the fluorescence intensity of coumarin-6 was evaluated with appropriately calibrated computerized image analysis, using “median density” as an analytic tool [Image Processing and Analysis in Java (Image J), National Institute of Mental Health, Bethesda, MD, USA]. The fluorescence intensity of coumarin-6 was measured in the range of 0–255 as the mean density, using Image J at the constant area (50 µm × 50 µm). The relative intensity indicates the value of a treated sample when the fluorescence intensity of an untreated sample is estimated as 1. A frozen section of eyeball was prepared, and the entire eye was scanned with fluorescence microscopy (BZ-9000, Keyence, Osaka, Japan) in order to obtain a schematic representation of the entire eye. 2.4. Evaluation of the rigidity of ssLip with atomic force microscopy (AFM) We used a commercial AFM apparatus (Nanoscope IIIa system controller, Digital Instruments Inc., Santa Barbara, CA, USA) with an Escanner possessing a maximum range of 10 µm × 10 µm × 2.5 µm. All images were captured in distilled water at room temperature with a silicon nitride probe (DNP-S20, Veeco Co., Ltd, nominal spring constant: 0.32 N/m). The scanning speed was optimized between 1.0 and 2.5 Hz depending on the scan size. All images were recorded by both height and amplitude modes, and they were analyzed in the height image mode. Surface-modified mica was used as the substrate for AFM observation. The mica was kept in a vacuum oven and was later prepared for surface modification using 3-aminopropyltriethoxysilane (AP) and N,N-diisopropylethylamine (DI) as reported by Thomson et al. [10]. Positively charged mica was used for adsorption between the mica and negatively charged liposomes. Freshly cleaved mica was incubated in a petri dish with AP (2 µl) and DI (1 µl) containing the top layer from the microcentrifuge tubes for 2 h. The liposomal suspension was diluted with distilled water, and the phospholipid concentration of liposomes was 20.4 µM. The surfacemodified mica and a quartz glass cell were set for the fluid tapping mode. Then, a suspension of ssLip was adsorbed onto the substrate surface, which was then washed three times with distilled water to
remove the non-adsorbed liposomes. AFM images were captured immediately after washing under suitable AFM conditions. The liposomes were visualized by both height and amplitude modes at a 10 µm × 10 µm scale after adsorption onto the substrate. With respect to the height of the liposomes, the depth of all liposomes was detected within a 10 µm × 10 µm mica surface area using AFM. The height (H) value represents the histogram mode by using the software included with this Nanoscope IIIa. Separately, the particle size (P) was measured using DLS method before the particles were adsorbed onto the substrate surface. These two parameters were introduced to describe the change in particle image height against the particle diameter as the rigidity of the ssLip (H/P) by the following equation: H/P = Mode of height of adsorbed ssLip (H) / Particle size of ssLip (P). 2.5. Cytotoxicity test using ocular cell lines The cytotoxicity of liposomes was evaluated using human conjunctival and corneal cell lines. Human conjunctival cells (Wong– Kilbourne derivative of Chang conjunctiva, clone 1 to 5c-41, CCL-20.2; American Type Culture Collection [ATCC], Manassas, VA, USA) were cultured under standard conditions (5% CO2, 95% humidified air, 37 °C) in medium-199 with Hank's salts (MP Biomedicals LLC) supplemented with 10% fetal bovine serum (GIBCO), 0.22% NaHCO3, 100 IU/ml streptomycin and 100 IU/ml penicillin (GIBCO). Immortalized human corneal epithelial cells (RIKEN Cell Bank, Tsukuba Science City, Japan) were cultured as described by Araki-Sasaki [11] in DMEM/Ham's F12 (1:1) supplemented with 10% FBS, 5 µg/ml insulin (Sigma), 0.1 µg/ml cholera toxin (List Biological Laboratories, Campbell, CA, USA), 0.5% DMSO, 10 ng/ml epidermal growth factor (Sigma), 100 IU/ml streptomycin and 100 IU/ml penicillin. The cell cultures were maintained at 37 °C, 5% CO2. The cytotoxicity of liposomes was determined by measuring the production of the yellow formazan product upon cleavage of 3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) by mitochondrial dehydrogenases in viable cells. The conjunctival cells and corneal cells were seeded at 3.15 × 104 cells/cm2 onto 96-well plates (Becton Dickinson, Franklin Lakes, NJ, USA). Cells were cultured for 4 days, and the culture medium was changed on alternate days (total 7 days). The culture medium was removed and washed twice with 200 µl of HBSS. The cells were then exposed to 100 µl of each sample. After 180 min incubation, the cells were washed three times with 150 µl HBSS. The cells were incubated with 20 µl of CellTiter 96® AQueous One Solution Reagent (Promega, Madison, WI, USA) composed of 317 µg/ml MTS in 100 µl of culture medium. After incubation in a CO2 incubator for 1 h, absorbance values were measured with a microplate reader (MTP 120, Corona Electric, Tokyo, Japan) at a wavelength of 492 nm. Background absorbance in cell-free wells was measured and subtracted from the measurement absorbance. The reference wavelength was 660 nm. A solution of 0.01% of benzalkonium chloride in HBSS–Hepes buffer served as a positive control. The percentage of cell viability was expressed as the percentage calculated by the following equation: % Cell viability = ABSsamples / ABScontrol × 100, where ABSsamples was the absorbance values of those wells exposed to the liposomal suspensions and ABScontrol was the absorbance values of those wells treated with HBSS–Hepes buffer.
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liposomes having different lipid formulations or particle sizes labeled with coumarin-6. The fluorescence of coumarin-6 was observed clearly in the retinal samples treated with EPC ssLip or DSPC ssLip. On the other hand, negligible fluorescence in the retina was found when EPC MLV, DSPC MLV or coumarin-6 dissolved in DMSO was dropped into the eyes. These fluorescence strengths were almost the same as those observed for the untreated eyeballs. To confirm the particle size effect of liposomal carriers, we have carried out the same experiment with the size-controlled larger liposomes; the average particle sizes were 200, 300, and 600 nm. The coumarin-6 intensity was decreased with increasing particle size (data are not shown). Since ssLips solely indicate positive results for coumarin-6 delivery to the posterior segment of the eyeball, time-course observation was carried out for these two liposomal samples. Fig. 2 shows the results of the time-course observation after EPC ssLip or DSPC ssLip administration. The fluorescence of coumarin-6 on the retinal images gradually increased with time, peaking 30 min after administration of either ssLip. The fluorescence thereafter decreased and was almost entirely disappeared at 180 min after administration. To compare the delivery efficiency to the retina of coumarin-6 vs. ssLips, the magnitude of green emission in the inner plexiform layer (IPL) of the retina was quantified using Image J software. The IPL seems to be a good target for evaluating retinal delivery, since it is located very close to the ganglion cell layer (GCL). The GCL contains retinal ganglion cells (RGC) and amacrine cells. RGC death is a common feature in many ophthalmic disorders, such as glaucoma, optic neuropathy and retinal vein occlusions [12]. Fig. 3 shows changes in the medium fluorescence density per pixel in the IPL for coumarin-6 entrapped liposomal systems. This analysis confirmed again that DSPC ssLip showed higher fluorescence emission in the retina, i.e., higher potency for drug delivery, than EPC ssLip, even though particle size and zeta potential were in a similar range. Although the Image J software could not
3. Results 3.1. Intraocular behavior of liposomes Epifluorescence microscopy was used to study the behavior of liposomes after they are dropped into the eye; for this purpose, frozen sections of sliced eye tissue were obtained as described in the Materials and methods section. Fig. 1 shows the retinal images observed for the samples taken 30 min after eyedrop administration of several types of
Fig. 1. Epifluorescence microscopic images of the retina 30 min after eyedrop administration. (a) Untreated, (b) contralateral eye, (c) coumarin-6 dissolved in DMSO, (d) EPC MLV, (e) DSPC MLV, (f) EPC ssLip, and (g) DSPC ssLip. GCL: ganglion cell layer, IPL: inner plexiform layer.
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Fig. 3. Changes in the accumulated fluorescence intensity in the inner plexiform layer (IPL) after eyedrop administration (mean ± SEM, n = 3). ⁎, P b 0.05; ⁎⁎⁎, P b 0.001, significantly different from EPC ssLip.
(P), which was measured using the DLS method before the particles were adsorbed onto the substrate surface, the H/P value may indicate the particle rigidity. We previously confirmed that size-controlled polystyrene particles almost uniformly showed H/P values of 1.0 [14]. As shown in Fig. 4, the H/P values of EPC ssLip and DSPC ssLip were 0.31 and 0.81, respectively. The higher ratio of the latter suggests that DSPC ssLip is more rigid than EPC ssLip. The phase transition temperatures of EPC and DSPC were reported to be −10 ± 5 °C and 55 °C, respectively [15]. This may explain the higher fluidity of EPC on liposomal membranes in comparison to DSPC.
Fig. 2. Epifluorescence microscopic images of the retina 5, 10, 30, 60 and 180 min after eyedrop administration. (a–e) EPC ssLip, and (f–j) DSPC ssLip. GCL: ganglion cell layer, IPL: inner plexiform layer.
accurately estimate the amount of coumarin-6 reaching the IPL, it could evaluate the relative potency with which ssLips of different lipid formulations delivered the hydrophobic compound to the IPL. 3.2. Rigidity of ssLip with AFM method AFM was used to evaluate the difference in rigidity between EPC ssLip and DSPC ssLip. Fig. 4 shows the AFM images of EPC ssLip and DSPC ssLip. Whereas EPC ssLip showed a flattened image, DSPC ssLip was spherical under the conditions of the AFM measurement. The height averages of EPC ssLip and DSPC ssLip, analyzed using AFM, were 31.3 and 124.6 nm, respectively. Several researchers have reported that the height of an AFM image is nearly identical to the actual particle size [13]. Since there is a good correlation between the mode of height and particle size, it can be assumed that the mode of height represents the length of the liposome perpendicular to the substrate surface. When the height of adsorbed particles on the AFM image (H) was divided by the particle size
Fig. 4. AFM images of ssLip adsorbed onto surface-modified mica (scale: 4 µm2 × 200 nm). (a) EPC ssLip, and (b) DSPC ssLip.
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3.3. Route of ssLip from ocular surface to retina To clarify ssLips' route to the retina after administration to the ocular surface, the entire eye was imaged 30 min after this administration to mice (Fig. 5). No fluorescence was observed in the optic nerve of the eye. Liposome-mediated fluorescence was observed on the surface of the cornea, as well as in the iris and the ciliary body. After preparing the frozen section of the eye, it was difficult to evaluate the coumarin-6 concentration precisely because the vitreous body and the aqueous humor involve high water content. The fluorescence of coumarin-6 was observed in anterior segment of the lens as shown in Fig. 5. This result may imply the distribution of coumarin-6 in the aqueous humor. 3.4. Cytotoxicity of liposomes Liposomes composed of phospholipids, which are cell components, are accepted as a biocompatible and nontoxic particulate drug carrier. However, some components tend to contribute to show cytotoxicity.
Fig. 6. Viability of conjunctival and corneal cells after exposure to liposomes as measured by MTS test (mean ± SEM, n = 8). (a) Conjunctival cells, and (b) corneal cells. BAK: 0.01% benzalkonium chloride in HBSS–Hepes buffer.
For example, positively charged liposomes containing stearylamine enhanced toxicity when their stearylamine content was increased [16]. It is also an acceptable claim that cytotoxicity depends strongly on the type of cells or tissues. Changes in the viability of conjunctival and corneal cells in the presence of liposomes are shown in Fig. 6. The viability of conjunctival and corneal cells remained unchanged when the cells came into contact with these liposomal suspensions. This confirmed very low cytotoxicity of these liposomes. Benzalkonium chloride solution (0.01%) significantly decreased viability, although this concentration of benzalkonium chloride is most commonly used in eyedrops. A quaternary ammonium moiety involved in the molecule causes morphologic disruption of the corneal epithelium and therefore induces apoptosis of Chang conjunctival cells [17]. However, the negatively charged liposomes used in this study are found to be highly biocompatible with low toxicity in ocular cells. 4. Discussion
Fig. 5. A schematic representation of the entire eye 30 min after administration of DSPC ssLip. (a) Iris, ciliary body, (b) cornea, (c) retina, and (d) optic nerve.
A number of review articles have discussed the delivery of drugs to the posterior segment of the eye via topical administration [4,5,18]. However, there has been no report in which a drug carrier system noninvasively targets the retina. One of the main problems of delivering drugs to the posterior segment of the eye is related to the presence of the corneal and conjunctival barriers. In our research to design carriers to better deliver drugs that are poorly absorbed by oral administration, we previously succeeded in the targeted absorption of peptide drugs by
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using liposomal formulations [19]. These experimental data encouraged us to carry out this trial in ocular delivery systems. As shown in Fig. 1, liposomes are able to deliver hydrophobic molecules into the retina. In comparing the different types of liposomes in size, ssLip was much more effective than MLV. We have reported the similar tendency in oral administration of peptide drugs with liposomal formulation. The tendency was elucidated by the result that ssLip could penetrate into the mucosal cells of rat intestine while such phenomenon could not be observed with larger liposomal particles (MLV) [6,19]. When the surface of ssLip was coated with mucoadhesive polymers such as chitosan, the absorption of drug was much increased. Based on these results we have estimated that the interaction between the drug carriers and biological surface in the intestinal tract such as mucous layer or mucosal cells is one of the important factors to control the delivery of drug administered. In ophthalmic drug delivery systems, nano-sized particles represent a state of matter characterized by higher bioadhesion [20] and greater surface area available for association between the cornea and conjunctiva. Kassem et al. reported that the mean residence time of drugs on the ocular surface increased as the particle size in the drug suspension decreased [21]. The longer residence time of ssLip compared to MLV may partly contribute to its behavior after administration to the ophthalmic surface. The longer retention of ssLip on the ocular surface may increase its association with the ocular surface tissues, the cornea and conjunctiva. The data shown in Fig. 2 confirmed that ssLip moved gradually to the retina within 30 min after administration. The disappearance of fluorescence at 180 min suggested two possible phenomena: clearance of the liposomal particles from the retina, and collapse of the liposomal structure and resultant diffusion of coumarin-6 molecules in the retina. Episcleral and choroidal circulation plays a significant role in clearing drugs from the retina after subconjunctival administration [22]. Amrite et al. reported that clearance of subconjunctivally administered 20 nm polystyrene particles can enter systemic circulation through the local intraocular circulation or following uptake by the conjunctival or episcleral blood vessels [23,24]. This disappearance of fluorescence might be attributed to the diffusion of ssLip itself or of coumarin-6 molecules to these periocular circulatory systems. The rigidity of liposomal particles is also an important factor in considering the drug delivery efficiency of liposomes. In this study, we confirmed that DSPC ssLip is more effective than EPC ssLip for delivering drugs to the retina. Rigid liposomes generally exhibit higher stability and are capable of maintaining entrapped substances. Muramatsu et al. reported that rigid liposomes containing insulin showed longer durations of blood glucose level reductions compared with fluid liposomes in oral administration, because highly rigid liposomes were stable at the surface of the intestinal mucosa and/or penetration through the mucosa occurred [25]. In contrast, insulin encapsulated in fluid liposomes showed high absorption of insulin in nasal administration according to a quick release of the drug from the liposomes [26]. The surface of the eye is covered by a continuous tear flow. The tear film consists of three layers: the adsorbed mucin layer, the middle aqueous layer and the superficial oily layer, with a total thickness of about 7–8 µm [27]. Although further investigation is required, liposome rigidity seems to be necessary in order to maintain the liposomal structure and entrapment of substances under these biological conditions. Absorption of liposomes after topical administration to the surface of the eye seemed to occur mainly via three routes: the systemic, corneal and non-corneal pathways [28]. Systemic delivery caused by nasolachrymal drainage did not contribute to the observed fluorescence in the retina, since no fluorescence was observed in the optic nerve of the eye or in the retina of the untreated contralateral eye. Corneal penetration was not thought to be a main route because fluorescence was observed only on the corneal surface. The cornea is comprised of three main layers: the outer epithelium, the stroma and the inner endothelium. The outer epithelium is lipophilic in nature, while the others are hydrophilic
[29]. The stroma and endothelium of the cornea, being hydrophilic, prevented the absorption of liposomes. The conjunctiva also plays an important role as a protective barrier on the ocular surface. However, the conjunctiva is not a strong barrier compared with the cornea. Its permeability was reported to be much greater than that of the cornea [30]. Drugs absorbed into the conjunctiva can enter the aqueous humor as well as the sclera, showing good access to the trabecular meshwork, iris root and pars plana [31]. Although further investigation is required, the liposomes may not penetrate through the sclera from the outer side of the eye. The delivery of liposomes to the posterior segment of the eye might mainly occur via the non-corneal pathways: liposomes may access through the tissues involvement trabecular meshwork, iris root and pars plana. 5. Conclusion The fluorescence emission of coumarin-6, a fluorescence dye used as a hydrophobic model compound, was obvious in the posterior segment of the eye after submicron-sized liposomes containing coumarin-6 were topically administered as eyedrops. The magnitude of fluorescence in the retina was closely related to the particle size and rigidity of the liposomes administered. Submicron-sized liposomes with rigid structures could be potential carriers for targeting the posterior segment of the eye. Epifluorescence microscopy of the entire eye revealed that the delivery route of liposomes to the posterior segment of the eye may not occur via corneal penetration or systemic delivery caused by nasolachrymal drainage. Those mechanisms will be further investigated in our next study using active pharmaceutical ingredients. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jconrel.2009.02.020. References [1] M. Colucciello, Intravitreal bevacizumab and triamcinolone acetonide combination therapy for exudative neovascular age-related macular degeneration: short-term optical coherence tomography results, J. Ocul. Pharmacol. Ther. 24 (1) (2008) 15–24. [2] E. Fattal, A. Bochot, Ocular delivery of nucleic acids: antisense oligonucleotides, aptamers and siRNA, Adv. Drug Deliv. Rev. 58 (11) (2006) 1203–1223. [3] T. Yasukawa, H. Kimura, Y. Tabata, Y. Ogura, Biodegradable scleral plugs for vitreoretinal drug delivery, Adv. Drug Deliv. Rev. 52 (1) (2001) 25–36. [4] S. Duvvuri, S. Majumdar, A.K. Mitra, Drug delivery to the retina: challenges and opportunities, Expert Opin. Biol. Ther. 3 (1) (2003) 45–56. [5] S.K. Sahoo, F. Dilnawaz, S. Krishnakumar, Nanotechnology in ocular drug delivery, Drug Discov. Today 13 (3–4) (2008) 144–151. [6] H. Takeuchi, Y. Matsui, H. Sugihara, H. Yamamoto, Y. Kawashima, Effectiveness of submicron-sized, chitosan-coated liposomes in oral administration of peptide drugs, Int. J. Pharm. 303 (1–2) (2005) 160–170. [7] I.P. Kaur, A. Garg, A.K. Singla, D. Aggarwal, Vesicular systems in ocular drug delivery: an overview, Int. J. Pharm. 269 (1) (2004) 1–14. [8] R.M. Hathout, S. Mansour, N.D. Mortada, A.S. Guinedi, Liposomes as an ocular delivery system for acetazolamide: in vitro and in vivo studies, AAPS Pharm. Sci. Tech. 8 (1) (2007) 1. [9] Y. Shen, J. Tu, Preparation and ocular pharmacokinetics of ganciclovir liposomes, Aaps J. 9 (3) (2007) E371–377. [10] N.H. Thomson, I. Collin, M.C. Davies, K. Palin, D. Parkins, C.J. Roberts, S.J.B. Tendler, P.M. Williams, Atomic force microscopy of cationic liposomes, Langmuir 16 (11) (2000) 4813–4818. [11] K. Araki-Sasaki, Y. Ohashi, T. Sasabe, K. Hayashi, H. Watanabe, Y. Tano, H. Handa, An SV40-immortalized human corneal epithelial cell line and its characterization, Invest. Ophthalmol. Vis. Sci. 36 (3) (1995) 614–621. [12] M. Shimazawa, Y. Inokuchi, Y. Ito, H. Murata, M. Aihara, M. Miura, M. Araie, H. Hara, Involvement of ER stress in retinal cell death, Mol. Vis. 13 (2007) 578–587. [13] K.A. Ramirez-Aguilar, K.L. Rowlen, Tip characterization from AFM images of nanometric spherical particles, Langmuir 14 (9) (1998) 2562–2566. [14] K. Nakano, Y. Tozuka, H. Yamamoto, Y. Kawashima, H. Takeuchi, A novel method for measuring rigidity of submicron-size liposomes with atomic force microscopy, Int. J. Pharm. 355 (1–2) (2008) 203–209. [15] K.M.G. Taylor, R.M. Morris, Thermal analysis of phase transition behaviour in liposomes, Thermochim. Acta 248 (2) (1995) 289–301. [16] K. Taniguchi, Y. Yamamoto, K. Itakura, H. Miichi, S. Hayashi, Assessment of ocular irritability of liposome preparations, J. Pharmacobiodyn. 11 (9) (1988) 607–611.
[17] M. De Saint Jean, C. Debbasch, F. Brignole, P. Rat, J.M. Warnet, C. Baudouin, Toxicity of preserved and unpreserved antiglaucoma topical drugs in an in vitro model of conjunctival cells, Curr. Eye Res. 20 (2) (2000) 85–94. [18] R.M. Mainardes, M.C. Urban, P.O. Cinto, N.M. Khalil, M.V. Chaud, R.C. Evangelista, M.P. Gremiao, Colloidal carriers for ophthalmic drug delivery, Curr. Drug Targets 6 (3) (2005) 363–371. [19] H. Takeuchi, H. Yamamoto, Y. Kawashima, Mucoadhesive nanoparticulate systems for peptide drug delivery, Adv. Drug Deliv. Rev. 47 (1) (2001) 39–54. [20] K. Yoncheva, E. Lizarraga, J.M. Irache, Pegylated nanoparticles based on poly (methyl vinyl ether-co-maleic anhydride): preparation and evaluation of their bioadhesive properties, Eur. J. Pharm. Sci. 24 (5) (2005) 411–419. [21] M.A. Kassem, A.A. Abdel Rahman, M.M. Ghorab, M.B. Ahmed, R.M. Khalil, Nanosuspension as an ophthalmic delivery system for certain glucocorticoid drugs, Int. J. Pharm. 340 (1–2) (2007) 126–133. [22] A.C. Amrite, H.F. Edelhauser, U.B. Kompella, Modeling of corneal and retinal pharmacokinetics after periocular drug administration, Invest. Ophthalmol. Vis. Sci. 49 (1) (2008) 320–332. [23] A.C. Amrite, H.F. Edelhauser, S.R. Singh, U.B. Kompella, Effect of circulation on the disposition and ocular tissue distribution of 20 nm nanoparticles after periocular administration, Mol. Vis. 14 (2008) 150–160. [24] A.C. Amrite, U.B. Kompella, Size-dependent disposition of nanoparticles and microparticles following subconjunctival administration, J. Pharm. Pharmacol. 57 (12) (2005) 1555–1563.
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[25] K. Muramatsu, Y. Maitani, T. Nagai, Dipalmitoylphosphatidylcholine liposomes with soybean-derived sterols and cholesterol as a carrier for the oral administration of insulin in rats, Biol. Pharm. Bull. 19 (8) (1996) 1055–1058. [26] K. Muramatsu, Y. Maitani, K. Takayama, T. Nagai, The relationship between the rigidity of the liposomal membrane and the absorption of insulin after nasal administration of liposomes modified with an enhancer containing insulin in rabbits, Drug. Dev. Ind. Pharm. 25 (10) (1999) 1099–1105. [27] H. Sasaki, K. Yamamura, K. Nishida, J. Nakamura, M. Ichikawa, Delivery of drugs to the eye by topical application, Prog. Retin. Eye Res. 15 (2) (1996) 583–620. [28] P.M. Hughes, O. Olejnik, J.E. Chang-Lin, C.G. Wilson, Topical and systemic drug delivery to the posterior segments, Adv. Drug Deliv. Rev. 57 (14) (2005) 2010–2032. [29] H.S. Huang, R.D. Schoenwald, J.L. Lach, Corneal penetration behavior of β-blocking agents II: assessment of barrier contributions, J. Pharm. Sci. 72 (11) (1983) 1272–1279. [30] K.M. Hamalainen, K. Kananen, S. Auriola, K. Kontturi, A. Urtti, Characterization of paracellular and aqueous penetration routes in cornea, conjunctiva, and sclera, Invest. Ophthalmol. Vis. Sci. 38 (3) (1997) 627–634. [31] S.B. Koevary, Pharmacokinetics of topical ocular drug delivery: potential uses for the treatment of diseases of the posterior segment and beyond, Curr. Drug Metab. 4 (3) (2003) 213–222.
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Journal of Controlled Release j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j c o n r e l
Design and evaluation of a liposomal delivery system targeting the posterior segment of the eye Kohei Hironaka a, Yuta Inokuchi b, Yuichi Tozuka a, Masamitsu Shimazawa b, Hideaki Hara b, Hirofumi Takeuchi a,⁎ a b
Laboratory of Pharmaceutical Engineering, Gifu Pharmaceutical University, Japan Laboratory of Biofunctional Evaluation, Molecular Pharmacology, Gifu Pharmaceutical University, Japan
a r t i c l e
i n f o
Article history: Received 17 October 2008 Accepted 17 February 2009 Available online 9 March 2009 Keywords: Liposomes Posterior segment of the eye Retina Atomic force microscopy Eyedrops
a b s t r a c t The purpose of this study was to evaluate the potential of submicron-sized liposomes (ssLips) as a novel system for delivering ocular drugs to the eye's posterior segment. Fluorescence emission of coumarin-6 formulated into ssLip was obvious in that segment in mice after eyedrop administration of the liposomal suspension. Such fluorescence was not observed after administration of either multilamellar vesicles or dimethyl sulfoxide (DMSO) solution containing the same amount of coumarin-6. The highest fluorescence of ssLip occurred 30 min after eyedrop administration, and all fluorescence disappeared after 180 min. The ssLip based on L-α-distearoyl phosphatidylcholine (DSPC ssLip) showed higher fluorescence emission in the retina than that based on egg phosphatidylcholine (EPC ssLip). These results confirmed that the magnitude of fluorescence in the retina was closely related to both liposome rigidity and particle size. Images of the entire eye showed that ssLip was delivered via the non-corneal pathway after administration. The liposomes tested in ocular cells showed little cytotoxicity. These results suggest that ssLip can be used to deliver drugs to the posterior segment of the eye. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The leading causes of vision impairment and blindness are posterior segment-related diseases including age-related macular degeneration, diabetic macular edema and endophthalmitis. Recently, pharmaceutical approaches to these diseases have used steroids and oligonucleotides [1,2]. These drugs are generally administered via invasive methods, such as intravitreal injections and subtenon injections, because noninvasive delivery of drugs is not available. However, repeated injections are associated with potential risks of complications, such as cataracts, vitreous hemorrhages and retinal detachment [3]. Moreover, patients may not comply with such regimens. Thus, there is a pressing need for noninvasive delivery systems targeting the posterior segment of the eye. Systemic administration is one possible way to obviate intravitreal or subtenon injection. One of the disadvantages of systemic administration to deliver drugs to the retina is its efficiency. A very small fraction of the systemically administered dose reaches the ocular tissues because the
⁎ Corresponding author. Gifu Pharmaceutical University, 5-6-1 Mitahora-higashi Gifu 502-8585, Japan. Tel: +81 58 237 8574; fax: +81 68 237 6524. E-mail address: [email protected] (H. Takeuchi). 0168-3659/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2009.02.020
blood-retinal barrier can prevent the drug from entering the retina [4]. Moreover, the doses that are needed in order to have a therapeutic effect via this route can lead to considerable side effects [5]. Topical administration with eyedrops is an alternative way to minimize side effects. However, corneal and conjunctival epithelia, along with tear film, serve as biological barriers to protect the eye from potentially harmful substances and drugs. Therefore, conventional eyedrop formulations usually cannot effectively overcome these barriers. The use of colloidal drug delivery systems, such as nanoparticles, nanoemulsions and liposomes, has received much attention as a way to enhance the bioavailability of drugs administered both systemically and topically. We have recently demonstrated that submicron-sized liposomes (ssLips) penetrated mucosal cells in rat intestine [6]. This finding encouraged us to try to use ssLip in an eyedrop formulation in this study. In ophthalmic therapy, many researchers have investigated the use of liposomes extensively. Liposomes reportedly can come into intimate contact with the ocular surfaces, thus working as barriers, and they can be used to protect drug molecules from metabolic enzymes present at the tear/corneal epithelium interface [7]. Hathout et al. reported that multilamellar liposomes containing acetazolamide were more efficient than acetazolamide solution in lowering intraocular pressure [8]. Shen and Tu reported that the ocular bioavailability of liposomal ganciclovir in liposomes in rabbits was 1.7-fold higher than that of a
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ganciclovir solution [9]. However, there have been no reports on drug delivery to the retina via eyedrops formulating a colloidal drug carrier such as liposomes. The purpose of this study was to evaluate the potential of ssLip as a novel system for delivering ocular drugs to the posterior segment of the eye, including the retina. The behavior of liposomes labeled with coumarin-6 as a fluorescence reagent was investigated after it was topically administered to mice via eyedrops. The cytotoxicity of the liposomes was also tested in vitro using ocular cells. 2. Materials and methods 2.1. Materials Egg phosphatidylcholine (EPC) and L-α-distearoyl phosphatidylcholine (DSPC) were purchased from Nippon Oil and Fats Co., Ltd. (Tokyo, Japan). Dicetyl phosphate (DCP) and cholesterol were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Coumarin-6 as a lipid marker and benzalkonium chloride were purchased from MP Biomedicals LLC (Illkirch, France). 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) was purchased from Nakalai Tesque (Kyoto, Japan). Hank's balanced salt solution (HBSS) was purchased from GIBCO BRL (Grand Island, NY, USA). Mica was purchased from Veeco Co., Ltd. (Tokyo, Japan). All other chemicals were commercial products of reagent grade. 2.2. Preparation of liposomes Multilamellar vesicles (MLV), composed of phospholipid (EPC or DSPC), DCP, and cholesterol at a molar ratio of 8:2:1, were prepared using the hydration method. The lipid mixture containing coumarin-6 was dissolved in a small amount of chloroform in a round-bottom flask and dried in a rotary evaporator under reduced pressure at 40 °C to form a thin lipid film. The film was dried in a vacuum oven overnight to ensure complete removal of the solvent. After addition of HBSS–Hepes buffer, lipid film was incubated in water bath at 70 °C for 30 s, and then vortexed for about 30 s. This cycle was repeated 5 times. The obtained MLV were incubated at 10 °C for 30 min. The ssLip was prepared using an extruder (LipoFast™-Pneumatic; Avestin, Inc., Ottawa, Canada) with a size-controlled polycarbonate membrane (0.1 µm membrane filter pore size; Whatman Japan KK, Tokyo, Japan). Extrusion was performed 41 times under nitrogen pressure (200 psi). The final phospholipid and coumarin-6 concentrations in the resultant liposomal suspension were 20.4 µmol/ml and 0.143 µmol/ ml, respectively. The coumarin-6 entrapment efficiency into ssLip was calculated as follows. The ssLip was obtained by ultrasonication of the MLV followed by filtering the ssLip through a cellulose acetate filter (0.8 µm). The filtered suspensions (0.5 ml) were dissolved in chloroform/methanol (1/1, 3 ml), and the coumarin concentration was measured with fluorometry (F3010, Hitachi, Tokyo, Japan) at an excitation wavelength of 490 nm and an emission wavelength of 520 nm. The entrapment efficiency percentage of ssLip was calculated by the following equation: % entrapment efficiency = Aflt / Aint × 100, where Aflt is the filtered amount of coumarin-6 in ssLip and Aint is the initial amount of coumarin-6 in ssLip. The ssLip particle size was measured with an aliquot of the particulate suspension diluted with a large amount of distilled water by the dynamic light scattering (DLS) method (Zetasizer, Malvern, Worcestershire, UK). The MLV particle size was measured by a laser diffraction size analyzer (LDSA-2400A; Tonichi Computer applications, Tokyo, Japan). The zeta potential of liposomes was measured using a laser Doppler method (Zetasizer, Malvern). Each batch was analyzed in triplicate. Both EPC and DSPC confirmed that more than 98% of coumarin-6 was entrapped into ssLip. As shown in Table 1, there were no differences in particle size, zeta potential or entrapment efficiency of coumarin-6 between the two types of phospholipids.
Table 1 Characterization of liposomes.
EPC MLV DSPC MLV EPC ssLip DSPC ssLip
Mean particle size (nm)
Zeta potential (mV)
5750 6450 125.3 105.4
− 97.5 − 101.1 − 62.9 − 66.2
2.3. Animal studies Un-anesthetized male adult ddY mice (Japan SLC, Hamamatsu, Japan) weighing 30–35 g were used. The mice were fed a regular diet. All experiments were approved and monitored by the Institutional Animal Care and Use Committee of Gifu Pharmaceutical University. A single dose of 3 µl of the liposomal formulation was dropped onto the surface of the left eye. The contralateral eye was used as the control and received no treatment. The mice were then sacrificed 5, 10, 30, 60 or 180 min after administration of the liposomal formulation. Both eyes were enucleated immediately and washed with excess amount of saline, and then fixed overnight in 4% paraformaldehyde at 4 °C. Fixed eyes were immersed in 20% sucrose for 24 h at 4 °C and embedded in optimal cutting temperature compound (Sakura Finetechnical Co., Ltd., Tokyo, Japan). The samples were then sliced with a cryostat (CM1850, Leica Instrument GmbH, Nussloch, Germany) into sections 10 µm thick and placed onto slides under a cover slip. The retinal images, taken at distances between 375 and 625 µm from the optic disc of frozen sections, were observed using epifluorescence microscopy (BX50; Olympus, Tokyo, Japan) with an attached CCD camera (DP30VW, Olympus) and fluorescence filters for coumarin-6 (U-WNIBA, Olympus). In the inner plexiform layer (IPL) at a distance between 475 and 525 µm (50 µm × 50 µm) from the optic disc, the fluorescence intensity of coumarin-6 was evaluated with appropriately calibrated computerized image analysis, using “median density” as an analytic tool [Image Processing and Analysis in Java (Image J), National Institute of Mental Health, Bethesda, MD, USA]. The fluorescence intensity of coumarin-6 was measured in the range of 0–255 as the mean density, using Image J at the constant area (50 µm × 50 µm). The relative intensity indicates the value of a treated sample when the fluorescence intensity of an untreated sample is estimated as 1. A frozen section of eyeball was prepared, and the entire eye was scanned with fluorescence microscopy (BZ-9000, Keyence, Osaka, Japan) in order to obtain a schematic representation of the entire eye. 2.4. Evaluation of the rigidity of ssLip with atomic force microscopy (AFM) We used a commercial AFM apparatus (Nanoscope IIIa system controller, Digital Instruments Inc., Santa Barbara, CA, USA) with an Escanner possessing a maximum range of 10 µm × 10 µm × 2.5 µm. All images were captured in distilled water at room temperature with a silicon nitride probe (DNP-S20, Veeco Co., Ltd, nominal spring constant: 0.32 N/m). The scanning speed was optimized between 1.0 and 2.5 Hz depending on the scan size. All images were recorded by both height and amplitude modes, and they were analyzed in the height image mode. Surface-modified mica was used as the substrate for AFM observation. The mica was kept in a vacuum oven and was later prepared for surface modification using 3-aminopropyltriethoxysilane (AP) and N,N-diisopropylethylamine (DI) as reported by Thomson et al. [10]. Positively charged mica was used for adsorption between the mica and negatively charged liposomes. Freshly cleaved mica was incubated in a petri dish with AP (2 µl) and DI (1 µl) containing the top layer from the microcentrifuge tubes for 2 h. The liposomal suspension was diluted with distilled water, and the phospholipid concentration of liposomes was 20.4 µM. The surfacemodified mica and a quartz glass cell were set for the fluid tapping mode. Then, a suspension of ssLip was adsorbed onto the substrate surface, which was then washed three times with distilled water to
remove the non-adsorbed liposomes. AFM images were captured immediately after washing under suitable AFM conditions. The liposomes were visualized by both height and amplitude modes at a 10 µm × 10 µm scale after adsorption onto the substrate. With respect to the height of the liposomes, the depth of all liposomes was detected within a 10 µm × 10 µm mica surface area using AFM. The height (H) value represents the histogram mode by using the software included with this Nanoscope IIIa. Separately, the particle size (P) was measured using DLS method before the particles were adsorbed onto the substrate surface. These two parameters were introduced to describe the change in particle image height against the particle diameter as the rigidity of the ssLip (H/P) by the following equation: H/P = Mode of height of adsorbed ssLip (H) / Particle size of ssLip (P). 2.5. Cytotoxicity test using ocular cell lines The cytotoxicity of liposomes was evaluated using human conjunctival and corneal cell lines. Human conjunctival cells (Wong– Kilbourne derivative of Chang conjunctiva, clone 1 to 5c-41, CCL-20.2; American Type Culture Collection [ATCC], Manassas, VA, USA) were cultured under standard conditions (5% CO2, 95% humidified air, 37 °C) in medium-199 with Hank's salts (MP Biomedicals LLC) supplemented with 10% fetal bovine serum (GIBCO), 0.22% NaHCO3, 100 IU/ml streptomycin and 100 IU/ml penicillin (GIBCO). Immortalized human corneal epithelial cells (RIKEN Cell Bank, Tsukuba Science City, Japan) were cultured as described by Araki-Sasaki [11] in DMEM/Ham's F12 (1:1) supplemented with 10% FBS, 5 µg/ml insulin (Sigma), 0.1 µg/ml cholera toxin (List Biological Laboratories, Campbell, CA, USA), 0.5% DMSO, 10 ng/ml epidermal growth factor (Sigma), 100 IU/ml streptomycin and 100 IU/ml penicillin. The cell cultures were maintained at 37 °C, 5% CO2. The cytotoxicity of liposomes was determined by measuring the production of the yellow formazan product upon cleavage of 3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) by mitochondrial dehydrogenases in viable cells. The conjunctival cells and corneal cells were seeded at 3.15 × 104 cells/cm2 onto 96-well plates (Becton Dickinson, Franklin Lakes, NJ, USA). Cells were cultured for 4 days, and the culture medium was changed on alternate days (total 7 days). The culture medium was removed and washed twice with 200 µl of HBSS. The cells were then exposed to 100 µl of each sample. After 180 min incubation, the cells were washed three times with 150 µl HBSS. The cells were incubated with 20 µl of CellTiter 96® AQueous One Solution Reagent (Promega, Madison, WI, USA) composed of 317 µg/ml MTS in 100 µl of culture medium. After incubation in a CO2 incubator for 1 h, absorbance values were measured with a microplate reader (MTP 120, Corona Electric, Tokyo, Japan) at a wavelength of 492 nm. Background absorbance in cell-free wells was measured and subtracted from the measurement absorbance. The reference wavelength was 660 nm. A solution of 0.01% of benzalkonium chloride in HBSS–Hepes buffer served as a positive control. The percentage of cell viability was expressed as the percentage calculated by the following equation: % Cell viability = ABSsamples / ABScontrol × 100, where ABSsamples was the absorbance values of those wells exposed to the liposomal suspensions and ABScontrol was the absorbance values of those wells treated with HBSS–Hepes buffer.
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liposomes having different lipid formulations or particle sizes labeled with coumarin-6. The fluorescence of coumarin-6 was observed clearly in the retinal samples treated with EPC ssLip or DSPC ssLip. On the other hand, negligible fluorescence in the retina was found when EPC MLV, DSPC MLV or coumarin-6 dissolved in DMSO was dropped into the eyes. These fluorescence strengths were almost the same as those observed for the untreated eyeballs. To confirm the particle size effect of liposomal carriers, we have carried out the same experiment with the size-controlled larger liposomes; the average particle sizes were 200, 300, and 600 nm. The coumarin-6 intensity was decreased with increasing particle size (data are not shown). Since ssLips solely indicate positive results for coumarin-6 delivery to the posterior segment of the eyeball, time-course observation was carried out for these two liposomal samples. Fig. 2 shows the results of the time-course observation after EPC ssLip or DSPC ssLip administration. The fluorescence of coumarin-6 on the retinal images gradually increased with time, peaking 30 min after administration of either ssLip. The fluorescence thereafter decreased and was almost entirely disappeared at 180 min after administration. To compare the delivery efficiency to the retina of coumarin-6 vs. ssLips, the magnitude of green emission in the inner plexiform layer (IPL) of the retina was quantified using Image J software. The IPL seems to be a good target for evaluating retinal delivery, since it is located very close to the ganglion cell layer (GCL). The GCL contains retinal ganglion cells (RGC) and amacrine cells. RGC death is a common feature in many ophthalmic disorders, such as glaucoma, optic neuropathy and retinal vein occlusions [12]. Fig. 3 shows changes in the medium fluorescence density per pixel in the IPL for coumarin-6 entrapped liposomal systems. This analysis confirmed again that DSPC ssLip showed higher fluorescence emission in the retina, i.e., higher potency for drug delivery, than EPC ssLip, even though particle size and zeta potential were in a similar range. Although the Image J software could not
3. Results 3.1. Intraocular behavior of liposomes Epifluorescence microscopy was used to study the behavior of liposomes after they are dropped into the eye; for this purpose, frozen sections of sliced eye tissue were obtained as described in the Materials and methods section. Fig. 1 shows the retinal images observed for the samples taken 30 min after eyedrop administration of several types of
Fig. 1. Epifluorescence microscopic images of the retina 30 min after eyedrop administration. (a) Untreated, (b) contralateral eye, (c) coumarin-6 dissolved in DMSO, (d) EPC MLV, (e) DSPC MLV, (f) EPC ssLip, and (g) DSPC ssLip. GCL: ganglion cell layer, IPL: inner plexiform layer.
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Fig. 3. Changes in the accumulated fluorescence intensity in the inner plexiform layer (IPL) after eyedrop administration (mean ± SEM, n = 3). ⁎, P b 0.05; ⁎⁎⁎, P b 0.001, significantly different from EPC ssLip.
(P), which was measured using the DLS method before the particles were adsorbed onto the substrate surface, the H/P value may indicate the particle rigidity. We previously confirmed that size-controlled polystyrene particles almost uniformly showed H/P values of 1.0 [14]. As shown in Fig. 4, the H/P values of EPC ssLip and DSPC ssLip were 0.31 and 0.81, respectively. The higher ratio of the latter suggests that DSPC ssLip is more rigid than EPC ssLip. The phase transition temperatures of EPC and DSPC were reported to be −10 ± 5 °C and 55 °C, respectively [15]. This may explain the higher fluidity of EPC on liposomal membranes in comparison to DSPC.
Fig. 2. Epifluorescence microscopic images of the retina 5, 10, 30, 60 and 180 min after eyedrop administration. (a–e) EPC ssLip, and (f–j) DSPC ssLip. GCL: ganglion cell layer, IPL: inner plexiform layer.
accurately estimate the amount of coumarin-6 reaching the IPL, it could evaluate the relative potency with which ssLips of different lipid formulations delivered the hydrophobic compound to the IPL. 3.2. Rigidity of ssLip with AFM method AFM was used to evaluate the difference in rigidity between EPC ssLip and DSPC ssLip. Fig. 4 shows the AFM images of EPC ssLip and DSPC ssLip. Whereas EPC ssLip showed a flattened image, DSPC ssLip was spherical under the conditions of the AFM measurement. The height averages of EPC ssLip and DSPC ssLip, analyzed using AFM, were 31.3 and 124.6 nm, respectively. Several researchers have reported that the height of an AFM image is nearly identical to the actual particle size [13]. Since there is a good correlation between the mode of height and particle size, it can be assumed that the mode of height represents the length of the liposome perpendicular to the substrate surface. When the height of adsorbed particles on the AFM image (H) was divided by the particle size
Fig. 4. AFM images of ssLip adsorbed onto surface-modified mica (scale: 4 µm2 × 200 nm). (a) EPC ssLip, and (b) DSPC ssLip.
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3.3. Route of ssLip from ocular surface to retina To clarify ssLips' route to the retina after administration to the ocular surface, the entire eye was imaged 30 min after this administration to mice (Fig. 5). No fluorescence was observed in the optic nerve of the eye. Liposome-mediated fluorescence was observed on the surface of the cornea, as well as in the iris and the ciliary body. After preparing the frozen section of the eye, it was difficult to evaluate the coumarin-6 concentration precisely because the vitreous body and the aqueous humor involve high water content. The fluorescence of coumarin-6 was observed in anterior segment of the lens as shown in Fig. 5. This result may imply the distribution of coumarin-6 in the aqueous humor. 3.4. Cytotoxicity of liposomes Liposomes composed of phospholipids, which are cell components, are accepted as a biocompatible and nontoxic particulate drug carrier. However, some components tend to contribute to show cytotoxicity.
Fig. 6. Viability of conjunctival and corneal cells after exposure to liposomes as measured by MTS test (mean ± SEM, n = 8). (a) Conjunctival cells, and (b) corneal cells. BAK: 0.01% benzalkonium chloride in HBSS–Hepes buffer.
For example, positively charged liposomes containing stearylamine enhanced toxicity when their stearylamine content was increased [16]. It is also an acceptable claim that cytotoxicity depends strongly on the type of cells or tissues. Changes in the viability of conjunctival and corneal cells in the presence of liposomes are shown in Fig. 6. The viability of conjunctival and corneal cells remained unchanged when the cells came into contact with these liposomal suspensions. This confirmed very low cytotoxicity of these liposomes. Benzalkonium chloride solution (0.01%) significantly decreased viability, although this concentration of benzalkonium chloride is most commonly used in eyedrops. A quaternary ammonium moiety involved in the molecule causes morphologic disruption of the corneal epithelium and therefore induces apoptosis of Chang conjunctival cells [17]. However, the negatively charged liposomes used in this study are found to be highly biocompatible with low toxicity in ocular cells. 4. Discussion
Fig. 5. A schematic representation of the entire eye 30 min after administration of DSPC ssLip. (a) Iris, ciliary body, (b) cornea, (c) retina, and (d) optic nerve.
A number of review articles have discussed the delivery of drugs to the posterior segment of the eye via topical administration [4,5,18]. However, there has been no report in which a drug carrier system noninvasively targets the retina. One of the main problems of delivering drugs to the posterior segment of the eye is related to the presence of the corneal and conjunctival barriers. In our research to design carriers to better deliver drugs that are poorly absorbed by oral administration, we previously succeeded in the targeted absorption of peptide drugs by
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using liposomal formulations [19]. These experimental data encouraged us to carry out this trial in ocular delivery systems. As shown in Fig. 1, liposomes are able to deliver hydrophobic molecules into the retina. In comparing the different types of liposomes in size, ssLip was much more effective than MLV. We have reported the similar tendency in oral administration of peptide drugs with liposomal formulation. The tendency was elucidated by the result that ssLip could penetrate into the mucosal cells of rat intestine while such phenomenon could not be observed with larger liposomal particles (MLV) [6,19]. When the surface of ssLip was coated with mucoadhesive polymers such as chitosan, the absorption of drug was much increased. Based on these results we have estimated that the interaction between the drug carriers and biological surface in the intestinal tract such as mucous layer or mucosal cells is one of the important factors to control the delivery of drug administered. In ophthalmic drug delivery systems, nano-sized particles represent a state of matter characterized by higher bioadhesion [20] and greater surface area available for association between the cornea and conjunctiva. Kassem et al. reported that the mean residence time of drugs on the ocular surface increased as the particle size in the drug suspension decreased [21]. The longer residence time of ssLip compared to MLV may partly contribute to its behavior after administration to the ophthalmic surface. The longer retention of ssLip on the ocular surface may increase its association with the ocular surface tissues, the cornea and conjunctiva. The data shown in Fig. 2 confirmed that ssLip moved gradually to the retina within 30 min after administration. The disappearance of fluorescence at 180 min suggested two possible phenomena: clearance of the liposomal particles from the retina, and collapse of the liposomal structure and resultant diffusion of coumarin-6 molecules in the retina. Episcleral and choroidal circulation plays a significant role in clearing drugs from the retina after subconjunctival administration [22]. Amrite et al. reported that clearance of subconjunctivally administered 20 nm polystyrene particles can enter systemic circulation through the local intraocular circulation or following uptake by the conjunctival or episcleral blood vessels [23,24]. This disappearance of fluorescence might be attributed to the diffusion of ssLip itself or of coumarin-6 molecules to these periocular circulatory systems. The rigidity of liposomal particles is also an important factor in considering the drug delivery efficiency of liposomes. In this study, we confirmed that DSPC ssLip is more effective than EPC ssLip for delivering drugs to the retina. Rigid liposomes generally exhibit higher stability and are capable of maintaining entrapped substances. Muramatsu et al. reported that rigid liposomes containing insulin showed longer durations of blood glucose level reductions compared with fluid liposomes in oral administration, because highly rigid liposomes were stable at the surface of the intestinal mucosa and/or penetration through the mucosa occurred [25]. In contrast, insulin encapsulated in fluid liposomes showed high absorption of insulin in nasal administration according to a quick release of the drug from the liposomes [26]. The surface of the eye is covered by a continuous tear flow. The tear film consists of three layers: the adsorbed mucin layer, the middle aqueous layer and the superficial oily layer, with a total thickness of about 7–8 µm [27]. Although further investigation is required, liposome rigidity seems to be necessary in order to maintain the liposomal structure and entrapment of substances under these biological conditions. Absorption of liposomes after topical administration to the surface of the eye seemed to occur mainly via three routes: the systemic, corneal and non-corneal pathways [28]. Systemic delivery caused by nasolachrymal drainage did not contribute to the observed fluorescence in the retina, since no fluorescence was observed in the optic nerve of the eye or in the retina of the untreated contralateral eye. Corneal penetration was not thought to be a main route because fluorescence was observed only on the corneal surface. The cornea is comprised of three main layers: the outer epithelium, the stroma and the inner endothelium. The outer epithelium is lipophilic in nature, while the others are hydrophilic
[29]. The stroma and endothelium of the cornea, being hydrophilic, prevented the absorption of liposomes. The conjunctiva also plays an important role as a protective barrier on the ocular surface. However, the conjunctiva is not a strong barrier compared with the cornea. Its permeability was reported to be much greater than that of the cornea [30]. Drugs absorbed into the conjunctiva can enter the aqueous humor as well as the sclera, showing good access to the trabecular meshwork, iris root and pars plana [31]. Although further investigation is required, the liposomes may not penetrate through the sclera from the outer side of the eye. The delivery of liposomes to the posterior segment of the eye might mainly occur via the non-corneal pathways: liposomes may access through the tissues involvement trabecular meshwork, iris root and pars plana. 5. Conclusion The fluorescence emission of coumarin-6, a fluorescence dye used as a hydrophobic model compound, was obvious in the posterior segment of the eye after submicron-sized liposomes containing coumarin-6 were topically administered as eyedrops. The magnitude of fluorescence in the retina was closely related to the particle size and rigidity of the liposomes administered. Submicron-sized liposomes with rigid structures could be potential carriers for targeting the posterior segment of the eye. Epifluorescence microscopy of the entire eye revealed that the delivery route of liposomes to the posterior segment of the eye may not occur via corneal penetration or systemic delivery caused by nasolachrymal drainage. Those mechanisms will be further investigated in our next study using active pharmaceutical ingredients. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jconrel.2009.02.020. References [1] M. Colucciello, Intravitreal bevacizumab and triamcinolone acetonide combination therapy for exudative neovascular age-related macular degeneration: short-term optical coherence tomography results, J. Ocul. Pharmacol. Ther. 24 (1) (2008) 15–24. [2] E. Fattal, A. Bochot, Ocular delivery of nucleic acids: antisense oligonucleotides, aptamers and siRNA, Adv. Drug Deliv. Rev. 58 (11) (2006) 1203–1223. [3] T. Yasukawa, H. Kimura, Y. Tabata, Y. Ogura, Biodegradable scleral plugs for vitreoretinal drug delivery, Adv. Drug Deliv. Rev. 52 (1) (2001) 25–36. [4] S. Duvvuri, S. Majumdar, A.K. Mitra, Drug delivery to the retina: challenges and opportunities, Expert Opin. Biol. Ther. 3 (1) (2003) 45–56. [5] S.K. Sahoo, F. Dilnawaz, S. Krishnakumar, Nanotechnology in ocular drug delivery, Drug Discov. Today 13 (3–4) (2008) 144–151. [6] H. Takeuchi, Y. Matsui, H. Sugihara, H. Yamamoto, Y. Kawashima, Effectiveness of submicron-sized, chitosan-coated liposomes in oral administration of peptide drugs, Int. J. Pharm. 303 (1–2) (2005) 160–170. [7] I.P. Kaur, A. Garg, A.K. Singla, D. Aggarwal, Vesicular systems in ocular drug delivery: an overview, Int. J. Pharm. 269 (1) (2004) 1–14. [8] R.M. Hathout, S. Mansour, N.D. Mortada, A.S. Guinedi, Liposomes as an ocular delivery system for acetazolamide: in vitro and in vivo studies, AAPS Pharm. Sci. Tech. 8 (1) (2007) 1. [9] Y. Shen, J. Tu, Preparation and ocular pharmacokinetics of ganciclovir liposomes, Aaps J. 9 (3) (2007) E371–377. [10] N.H. Thomson, I. Collin, M.C. Davies, K. Palin, D. Parkins, C.J. Roberts, S.J.B. Tendler, P.M. Williams, Atomic force microscopy of cationic liposomes, Langmuir 16 (11) (2000) 4813–4818. [11] K. Araki-Sasaki, Y. Ohashi, T. Sasabe, K. Hayashi, H. Watanabe, Y. Tano, H. Handa, An SV40-immortalized human corneal epithelial cell line and its characterization, Invest. Ophthalmol. Vis. Sci. 36 (3) (1995) 614–621. [12] M. Shimazawa, Y. Inokuchi, Y. Ito, H. Murata, M. Aihara, M. Miura, M. Araie, H. Hara, Involvement of ER stress in retinal cell death, Mol. Vis. 13 (2007) 578–587. [13] K.A. Ramirez-Aguilar, K.L. Rowlen, Tip characterization from AFM images of nanometric spherical particles, Langmuir 14 (9) (1998) 2562–2566. [14] K. Nakano, Y. Tozuka, H. Yamamoto, Y. Kawashima, H. Takeuchi, A novel method for measuring rigidity of submicron-size liposomes with atomic force microscopy, Int. J. Pharm. 355 (1–2) (2008) 203–209. [15] K.M.G. Taylor, R.M. Morris, Thermal analysis of phase transition behaviour in liposomes, Thermochim. Acta 248 (2) (1995) 289–301. [16] K. Taniguchi, Y. Yamamoto, K. Itakura, H. Miichi, S. Hayashi, Assessment of ocular irritability of liposome preparations, J. Pharmacobiodyn. 11 (9) (1988) 607–611.
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