In a number of review articles, the topical administration of drugs to the posterior segment of the eye is discussed.
7,8,16 However, there have been few reports in which a drug carrier system noninvasively targets the retina. One of the primary problems about delivering drugs to the posterior segment of the eye is the presence of corneal and conjunctival barriers. As described previously, ssLips have potential for delivering ophthalmic drugs to the posterior segment of the eye, and the rigidity of liposomal particles is an important factor in considering delivery efficiency to the retina.
15 In the present study, we focused on the effect of particle properties—e.g., particle size, surface charge, constituents—on intraocular behavior after eyedrop administration in mice, rabbits, and monkeys. To compare the delivery efficiency of these ssLips to the retina, the magnitude of green emission in the IPL of the retina was quantified using ImageJ software. The IPL seems to be a good target for evaluating retinal delivery because it is located very close to the GCL. Retinal ganglion cell death is a common feature in many ophthalmic disorders, such as glaucoma, optic neuropathy, and retinal vein occlusions.
17
Particle size is the most important factor in improving the drug delivery efficiency of carrier particles. As shown in
Figure 2, delivery efficiency of coumarin-6 was extensively improved by reducing liposomal particle size to the submicron order. In ophthalmic delivery systems, nano-sized particles represent a greater surface area available for association between the cornea and the conjunctiva.
18 Kassem et al.
19 have reported that the mean residence time of drugs on the ocular surface increases as the particle size in the drug suspension decreases. The extensive delivery efficiency to the retina of coumarin-6 was attributed to the ability of liposomes to associate with the ocular surface tissues, partly because of their prolonged retention property on the ocular mucosa. The solubility of coumarin-6 to distilled water is extremely low; hence, DMSO solution was used as a control because its coumarin-6 concentration is similar to that of the liposomal formulation. No fluorescence was observed for the DMSO solution containing coumarin-6, in contrast to the liposomal formulations.
15 Therefore, coumarin-6 molecules dissolved in DMSO solution cannot deliver to the retina as a molecular state.
As shown
Figure 3A, no fluorescence was observed in the retina after eyedrop administration of polystyrene particles. The lipid emulsions showed negligible emission of coumarin-6 after administration, whereas ssLips showed remarkable emission intensity. The delivery efficiency was different among those colloidal systems, even if both their particle size and zeta potential were approximately controlled as 110 nm and −50 mV, respectively. Given that polystyrene particles contain covalently bonded FITC, the absence of fluorescence in the retina suggested that polystyrene particles themselves could not reach the retina in our experiment. Amrite et al.
20 reported that 20-nm polystyrene particles did not permeate the sclera-choroid-RPE in 24 hours. They also reported that intraocular tissues such as the retina and the vitreous did not have any quantifiable uptake of the polystyrene particles (particle size: 20, 200, 2000 nm) after subconjunctival administration.
21 Particle size is definitely a dominant parameter for effective delivery; however, another factor should be considered when choosing a colloidal carrier for retinal delivery.
For the first step of the delivery, the colloidal particle should associate with the surface of the eye. The affinity between colloidal particles and cells may be a considerable factor in delivering drugs to the retina. It is well known that liposomes are composed of phospholipid, such as cell membrane (
Fig. 1), and they show good affinity with the ocular barrier, such as the cornea and the conjunctiva.
22 Lipid emulsions are exploited commercially as a vehicle to improve the ocular bioavailability of lipophilic drugs.
23,24 Naveh et al.
25 noted that the intraocular pressure–reducing effect of a topically administered dose of pilocarpine-loaded lipid emulsions is prolonged compared with that of generic pilocarpine. Calvo et al.
26 observed an improvement in indomethacin ocular bioavailability when the drug is incorporated in a lipid emulsion compared with commercial aqueous drops after topical application into the rabbit eye. Liposomes and lipid emulsion may have higher affinity than polystyrene particles. The lower affinity of polystyrene particles to cells may be the reason polystyrene particles do not show any fluorescence in the retina. Although both liposomes and lipid emulsions may have good affinity toward cells, coumarin-6 was not delivered effectively to the retina after administration of lipid emulsions (
Fig. 3B). The excellent delivery efficiency of ssLips to the retina was not solely explained by the affinity to cells. Lipid emulsions are composed of an oil core surrounded by a phospholipid monolayer, whereas liposomes are composed of phospholipid bilayers. We assumed that the structural differences altered the stability of those colloidal particles and the release profile of hydrophobic material. The delivery efficiency of coumarin-6 might be attributed to the structural difference of liposome and lipid emulsion; in this experiment, the phospholipid bilayers of liposomes might have been better surroundings for coumarin-6 than the phospholipid monolayer of lipid emulsions.
As shown in
Figure 4, the delivery efficiency of coumarin-6 from liposomes decreased with an increase in liposomal cholesterol content. Cholesterol usually acts as a stabilizer for liposome formulation. In fact, the hardness of liposomes increased with increasing cholesterol content with which the unsaturated phospholipid (e.g., egg phosphatidylcholine [EPC]) was used as a lipid component of liposomes (EPC/DCP/cholesterol). However, cholesterol might have acted as a plasticizer in our formulation (DSPC/DCP/cholesterol) using saturated phospholipid of DSPC as a lipid component of liposomes. This trend has already been reported by Utsumi et al.
27 In addition, we previously reported that atomic force microscopy images showed that DSPC/SA/cholesterol liposomes changed from spheres to ovals on mica surfaces with an increase in cholesterol content, indicating the decrease of liposomal rigidity with an increase in cholesterol content.
28 The rigidity of DSPC/DCP/cholesterol liposomes may decrease with an increase in cholesterol content. We have reported that the efficiency of coumarin-6 delivery to the retina is higher for DSPC/DCP/cholesterol liposomes than for EPC/DCP/cholesterol liposomes.
15 These results supported the idea that the lower rigidity of liposomes is disadvantageous for retinal delivery.
Several researchers have reported that the surface charge of liposomes affects the pharmacokinetics of drugs entrapped in liposomes.
9,10 Law et al.
9 reported that the absorption rate of acyclovir in positively charged liposomes is slower than the absorption rate of acyclovir in negatively charged liposomes. Hathout et al.
10 reported that acetazolamide-entrapped positively charged MLV lowered intraocular pressure more than negatively charged MLV. In contrast to expectations, there were no significant differences in intraocular behavior among negatively, neutrally, and positively charged ssLips (
Fig. 5). The positively charged liposome (DSPC/SA/cholesterol, 8:0.2:1) and the neutral liposome (DSPC/cholesterol, 8:1) exhibited an effective transfer of coumarin-6 to the retina. Although further investigations are required using several types of charged liposomes, ssLips with different surface charges might be potential carriers for retinal delivery.
Mouse eyes are very small, and their ocular barriers may be weaker than those of larger animals. The mean thickness of the cornea, the main absorption barrier, in mice, rats, and rabbits is approximately 110 μm, 160 μm, and 350 μm, respectively.
29 Therefore, experiments using other animals are required to confirm the penetration of coumarin-6 to the retina. As shown in
Figures 7 and
8, fluorescence emission of coumarin-6 was clearly observed in the retinas of rabbits. The rabbit eye is approximately 70% to 80% the size of the human eye in terms of axial length, diameter, corneal thickness, scleral thickness at the limbus, scleral thickness at the equator, and scleral surface area.
30 In addition, the fluorescence emission of coumarin-6 was also observed in the retinas of monkeys (
Fig. 9). Eyes of mammals such as rabbits, pigs, and monkeys are similar to those of humans. For these reasons, our findings are valuable for designing ocular drug delivery systems that target the human retina.
Data shown in
Figure 8 confirm that ssLip moved gradually to the retina within 60 minutes of administration. The disappearance of fluorescence at 360 minutes suggests 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 circulations play a significant role in clearing drugs from the retina after subconjunctival administration.
31 Amrite et al.
20,21 reported that clearance of subconjunctivally administered 20-nm polystyrene particles can enter systemic circulation through the local intraocular circulation after uptake by the conjunctival or episcleral blood vessels. This disappearance of fluorescence might be attributed to the diffusion of ssLip itself or of coumarin-6 molecules to these periocular circulatory systems.
Absorption of liposomes after topical administration to the surface of the eye is assumed to occur primarily through three routes: systemic, corneal, and noncorneal pathways.
32 However, negligible fluorescence in the retina of the contralateral eye (
Figs. 7B,
7D,
7F) exhibited no contribution of systemic delivery from nasolacrimal drainage. It may be that liposomes are delivered to the retina by way of a transscleral pathway. As shown in
Figure 2, the fluorescence emission in OPL proportionally increased with a decrease in particle size that corresponded well to the results shown in IPL. If liposomes were delivered by transscleral pathway, the concentration gradient of coumarin-6 from the scleral side to vitreous side of the retina may be observed. Additional evidence exists for the inability of small 100-nm liposomes small PLGA nanoparticles to permeate the sclera.
33 Therefore, the movement of intact liposomes to the retina by transscleral pathways is unlikely. Based on the entire eye image after the administration of ssLip, we previously considered that the delivery of liposomes to the posterior segment of the eye may occur primarily by noncorneal pathways—liposome access through the tissues involving the trabecular meshwork, iris root, and pars plana.
15 However, we cannot rule out the possibility of the transscleral route. Further investigations will be needed in the future. The coumarin-6 concentration gradient from the iris and ciliary body to the optic disc was observed in the retinal flat-mount image (
Fig. 6B). All four petal-shaped images exhibit similar fluorescence images depicting a fluorescence gradient from the ocular surface to the retina. This finding suggests that liposome-mediated fluorescence was distributed homogeneously in the dorsal, ventral, temporal, and nasal retina after eyedrop administration of ssLip.
In the animal study using mice, rabbits, and monkeys, fluorescence emission of coumarin-6 was obvious in the retina when submicron-sized liposomes were topically administered as eyedrops. The magnitude of fluorescence in the retina was closely related to the particle size of liposomes. Polystyrene particles and lipid emulsions showed an insufficient effect compared with that of liposomes on retinal delivery, even if particle size and zeta potential were in a similar range. The liposomal surface charge was not affected by the intraocular behavior of liposomes by eyedrop administration. Epifluorescence microscopy of the retinal flat-mount image revealed that the delivery of liposomes to the retina might have occurred by diffusion from ocular surface involvement to the iris ciliary body side. These mechanisms will be investigated further with active pharmaceutical ingredients.
Supported by a Grant-in-Aid (21390011) from the Ministry of Education, Culture, Sports, Sciences and Technology (Monbukagakusho) of Japan.