Many sustained intraocular drug delivery methods have been developed as alternatives to implantation. Ocular drug delivery systems using particulates have been developed, which provide sustained release with high target specificity in the form of microspheres and microcapsules with diameters of 1–1000 µm, as well as nanospheres and nanocapsules with diameters of less than 1 µm [54
]. Drugs can be incorporated into biodegradable polymers to form either a matrix system or a reservoir system [58
]. In a matrix system, the drugs and polymer are combined and the drug is released through diffusion from the polymer matrix with simultaneous polymer degradation. This system is used for micro-and nanospheres. The reservoir-type system involves encapsulating drugs within polymeric shells and is the system used for biodegradable micro- and nanocapsules [58
]. Some of the commonly used synthetic biodegradable polymers are the aliphatic polyesters such as PLA, PGA, PLGA and poly(caprolactone) [56
]. These polymers are appropriate for controlled-release applications because they are nontoxic, nonimmunogenic and degrade through enzymatic reactions and hydrolysis to natural metabolic products over a period of months to years [60
]. Drug-release profiles can be modified through variations in polymer molecular weights and copolymer formulations [57
]. An emulsion–diffusion process is used to encapsulate drugs in micro- and nanocapsules [62
], while solvent evaporation is used to prepare micro- and nanospheres [64
] using an oil-in-water emulsion for hydrophobic drugs or an oil-in-oil emulsion for improved encapsulation efficiency for hydrophilic drugs [58
]. The particulates are suspended in a carrier solution to enable ocular injection. Intravitreal injections can potentially impair vision due to clouding. However, microspheres larger than 2 µm tend to settle out owing to gravity and nanoparticulates diffuse quickly and localize within ocular tissues [7
Polymeric microspheres have been used to target the RPE. Moritera et al.
studied the use of surface-modified polymeric microspheres to localize drugs to the RPE [65
]. Phagocytosis by RPE was tracked by incorporating fluorescent dye into PLA microspheres with the rate of phagocytosis enhanced with gelatin-precoating as compared with bare microspheres. Kimura et al.
used polymeric microspheres formulated using l
-lactic acid and dl
-lactic acid and their copolymers loaded with a fluorescent dye, rhodamine 6GX, to evaluate drug targeting to the RPE [66
]. Intracellular dye release occurred following phagocytosis and could be controlled by varying the polymer formulation of the microspheres. Tuovinen et al.
studied targeting drugs to the RPE using microparticles (11 µm in diameter) produced with starch acetate, which degrades more slowly than native starch [67
]. These microparticles could be phagocytosed and degraded within the RPE.
Nanospheres have also been used to target the RPE for sustained drug delivery. Sakurai et al.
studied the intraocular kinetics of nanospheres and found that polystyrene nanospheres containing fluorescein (2 µm in diameter) were detectable in the retina, vitreous and trabecular meshwork more than 1 month following an intravitreal injection in vivo
in rabbits [68
]. Bourges et al.
showed the feasibility of targeting the retina and the RPE using a single intravitreal injection of polylactide nanoparticles loaded with the dye, rhodamine 6G and Nile Red, which quickly accessed the retina and were observed for 4 months postinjection [54
]. Kim et al.
used human serum albumin nanoparticles to track the movement of intravitreally injected nanoparticles as a function of surface charge and retinal injury [69
]. Anionic nanoparticles traversed the collagen fibrils of the vitreous more readily than the cationic nanoparticles, showing potential as drug delivery vehicles for the subretinal space and the RPE. Müller cells take up the nanoparticles, possibly playing a key role in retinal penetration. Gaudana et al.
reported that ligands, such as folate and biotin, attached to the surface of steroidal nanoparticles, can increase uptake by the RPE [6
Steroids such as budesonide and dexamethasone have been tested in polymeric nano- and microparticles for sustained drug delivery. Kompella et al.
determined that nano- and microparticles containing budesonide, a corticosteroid, could inhibit VEGF expression in vitro
in a RPE cell line (ARPE-19) [70
]. PLA nano-(345 nm) and microparticles (3.6 µm) containing budesonide were subconjunctivally injected in rats and were able to sustain retinal levels of budesonide compared with the steroid solution alone. Loftsson et al.
evaluated delivering steroids to the retina in rabbits by topical application of a low viscosity aqueous suspension, which contained dexamethasone/γ-cyclodextrin microparticles with a mean diameter of 20.4 µm, and achieved vitreal and retinal steroid concentrations comparable to levels observed 1 month after an intravitreal injection [71
]. Gomez-Gaete et al.
prepared dexamethasone-loaded PLGA nanoparticles (230 nm) and optimized the solvent evaporation process to produce particles with the highest drug entrapment [72
]. Gomez-Gaete et al.
then devised a new drug vehicle for the intravitreal delivery of dexamethasone, called Trojan particles, which were formed by spray drying 1,2-dipalmitoyl-SN
-glycero-3-phosphocholine, hyaluronic acid and different concentrations of dexamethasone-loaded PLGA nanoparticle suspensions [73
]. The Trojan particles, which are spherical, hollow and have surface irregularities due to encapsulated nanoparticles, permit slower in vitro
drug release owing to the protection provided by the excipient matrix.
The efficacy of encapsulating antiviral drugs such as ganciclovir and acyclovir into polymeric micro- and nanospheres has been presented. Veloso et al.
used ganciclovir-loaded PLGA microspheres (300–500 µm in diameter) in rabbits inoculated with human CMV and were able to control the progression of fundus disease using an intravitreal injection of 10 mg of microspheres (89.77 µg ganciclovir/mg) suspended in 0.1 ml of 2% hydroxypropylmethylcellulose [74
]. Herrero-Vanrell et al.
prepared ganciclovir-loaded PLGA microspheres (300–500 µm) by dispersing ganciclovir in fluorosilicone oil, which enabled a high ganciclovir loading (95%) [75
]. Microsphere sterilization using γ-radiation at a dose of 2.5 megarads did not affect the drug-release kinetics. Merodio et al.
studied the ocular toxicity of an intravitreal injection in rats of ganciclovir-loaded bovine serum albumin nanoparticles, which were well tolerated in vivo
and did not result in any inflammatory reactions based on histological evaluation [76
]. Albumin nanoparticles contain a significant number of charged amino acids, which enable the adsorption of ganciclovir or particles that carry a negative charge such as oligonucleotides, as shown in a study by Irache et al.
]. Duvvuri et al.
investigated drug entrapment, surface morphology, particle size analysis and drug release from ganciclovir-loaded PLGA microspheres using various polymer blends, showing how blends can modify drug release for controlled delivery systems [78
]. Duvvuri et al.
presented empirical equations to describe drug release from ganciclovir-loaded PLGA microspheres and developed a thermogelling polymer solution to carry the dispersed microspheres for sustained delivery [79
]. Duvvuri et al.
developed a sustained-release formulation of ganciclovir-loaded PLGA microspheres in a thermogelling PLGA–poly(ethylene glycol) (PEG)–PLGA solution that was tested in vivo
by intravitreal administration in a microdialysis rabbit model [80
]. The microspheres in the thermogel polymer solution could maintain mean ganciclovir levels of 0.8 µg/ml for 14 days as compared with 54 h with direct injections. Martinez-Sancho et al.
prepared PLGA microspheres loaded with vitamin A palmitate (10–80 mg) and acyclovir (40–80 mg) with in vitro
drug release sustained for 49 days using an optimal formulation of acyclovir 40 mg, vitamin A palminate 80 mg and polymer 400 mg [82
]. Cortesi et al.
used spray drying to encapsulate acyclovir in polyacrylic microparticles that exhibited a controlled drug-release profile [83
Pharmaceutical agents for the treatment of PVR have been incorporated into injectable particulates and tested for sustained delivery [56
]. Moritera et al.
studied the drug-release kinetics of microspheres (50 µm in diameter) prepared with polymers of PLA or copolymers of PGA and PLA containing 5-fluorouracil (5-FU), a potent inhibitor of fibroblast proliferation [84
]. Copolymers increased the rate of drug release over homopolymers. Drug-release rates were accelerated with lower molecular-weight polymers or a copolymer matrix. In vivo
studies in rabbits found increased microsphere clearance in vitrectomized eyes and electroretinograms, and histological study demonstrated no retinal toxicity. Moritera et al.
investigated PLA microspheres loaded with adriamycin for the treatment of PVR [85
]. In an experimental rabbit model, a single injection of microspheres containing adriamycin 10 µg decreased traction retinal detachment from 50% in controls to 10%, and was shown to be nontoxic to the retina by electroretinography and histological studies. However, retinal necrosis and detachment were observed with an injection of the same amount of free adriamycin, showing the potential for reduced toxicity through drug incorporation into biodegradable polymers. Giordano et al.
showed that PLGA (50:50) microspheres loaded with retinoic acid could provide sustained release for 40 days in vitro
and reduced the incidence of traction retinal detachment in a rabbit model of PVR after 2 months following a single intravitreal injection [86
]. Peyman et al.
tested the drug kinetics of microspheres formulated from copolymers of PLA and PGA (85:15), which incorporated cytosine arabinoside or 5-FU [87
]. The drug-loaded microspheres were injected intravitreally in primates with both drugs detectable at 11 days postinjection and exhibiting similar rates of drug clearance. Yeh et al.
prepared 3-µm microparticles using PLGA loaded with 5-FU and optimized the formulation to achieve an in vitro
sustained release of 5-FU for 21 days with a delivery rate of 0.4 µg 5-FU/mg particles/day [88
De Kozak et al.
investigated the efficacy of incorporating tamoxifen, a nonsteroidal estrogen-receptor modulator, into PEG-coated nanoparticles for the treatment of experimental autoimmune uveoretinitis. Intravitreal injection in a rat model performed 1–2 days before expected disease onset in controls significantly inhibited the disease owing to a shift in the immune response from a Th1 to a Th2-type response [89
]. He et al.
evaluated cyclosporine-loaded PLGA microspheres, 50 µm in diameter, for the treatment of uveitis [90
]. Drug release was monitored following intravitreal injections in healthy rabbits, maintaining therapeutic concentrations for at least 65 days in the choroid–retina and iris–ciliary body. Sakai et al.
investigated the iv. administration of PLA nanoparticles loaded with β-methasone phosphate and tagged with rhodamine to target experimental autoimmune uveoretinitis induced with S-antigen peptide in a rat model [91
]. The nanoparticles accumulated in the retina and choroid within 3 h and remained for 7 days postinjection, resulting in a reduction in the ocular infiltration of activated T cells and macrophages, as well as reduced hypertrophy of Müller cells. Barcia et al.
tested the short- and long-term efficacy of dexamethasone-loaded PLGA microspheres (20–53 µm in diameter) to reduce ocular inflammation in a rabbit model of uveitis elicited by intravitreal lipopolysaccharide injection [92
]. Both the short-term (15 days in length) and the long-term study (33 days in length) demonstrated reduced inflammation by clinical evaluation, electroretinography and histopathologic evaluation.
A novel approach for scavenging reactive oxygen species prominent in retinal degenerative diseases was presented by Chen et al.
] and reviewed by Edelhauser et al.
]. Cerium oxide nanoparticles (CeO2
, nanoceria particles), which are nontoxic, nonimmunogenic and protective at a very low dosage, provided protection in vivo
using a light-damage animal model. In this case, these rare earth particulates are not the carrier of a specific drug, but the therapeutic agent itself.
Micro- and nanoparticles have potential in the field of gene therapy by functioning as nonviral vectors to enable cellular penetration, guard against degradation and maintain sustained delivery. Panyam et al.
demonstrated that PLGA nanoparticles could escape the endo–lysosomal compartment and prevent degradation by lysosomal nucleases, a quality necessary for a drug delivery vehicle [96
]. The method of escape involves a reversal of the nanoparticle’s surface charge from anionic to cationic owing to the acidic environment of the endo–lysosomal compartment. This enables the nanoparticle to exit into the cytosol by interacting with the endo–lysosomal membrane. Endo–lysosomal escape makes PLGA nanoparticles an attractive delivery vehicle for macromolecules, such as DNA and low-molecular-weight drugs such as dexamethasone. Bejjani et al.
explored the use of PLA and PLGA nanoparticles as vectors for gene transfer to a bovine and a human ARPE-19 cell line [97
]. The plasmids employed were green fluorescent protein for expression within the cytoplasm or red nuclear fluorescent protein for expression within the nucleus. Intravitreal injections in vivo
in a rat model concluded that PLGA could successfully sequester and internalize plasmids, resulting in gene expression in RPE detectable 48 h postinjection and maintained for 8 days. Mo et al.
used human serum albumin nanoparticles loaded with the CuZn superoxide dismutase (SOD1
) gene for in vitro
transfection studies using human ARPE-19 cells [98
]. The gene-loaded nanoparticles had a transfection efficiency of 80%, a fivefold increase in SOD1
expression over untreated cells and no cytotoxicity. In vivo
studies employing an intravitreal injection in a mouse model resulted in detectable fusion protein at 48 h, while levels were undetectable in the control group.
Another therapeutic approach in the treatment of ocular disease is the inhibition of gene expression using antisense oligonucleotides, aptamers and siRNA [99
]. Aukunuru et al.
showed that nanoparticles formulated using a PLGA (50:50) copolymer could deliver VEGF antisense oligonucleotide to the human ARPE-19 cell line, and inhibit VEGF secretion and mRNA expression [102
]. In a study performed by Carrasquillo et al.
] and summarized by Moshfeghi and Peyman [104
], the anti-VEGF RNA aptamer (EYE001, Macugen®
, OSI Pharmaceuticals, NY, USA) was incorporated into PLGA microspheres to develop a sustained-release inhibition of VEGF for the treatment of neovascular AMD. The aptamer-loaded PLGA microspheres could deliver 2 µg/day over a 20-day period, effectively inhibiting VEGF-induced cell proliferation in human umbilical vein endothelial cells. The potential for using the PLGA microspheres for transscleral drug delivery to the choroid and retina was assessed in vitro
using a device in which harvested rabbit sclera was mounted and drug permeation could be measured. Spectrophotometry was used to verify aptamer transfer across the sclera for the 6-day test period. Singh et al.
presented a novel application using an iv. injection of surface-functionalized PLGA nanoparticles to target neovascular tissue for gene delivery of anti-VEGF intraceptor, an intracellular VEGF inhibitor, in a laser-induced, rodent model of choroidal neovascularization [105
]. Anti-VEGF intraceptor expression was increased in retinal vascular endothelial cells, photoreceptor outer segments and RPE cells using iv. administration of nanoparticles functionalized with either transferrin, arginine–glycine–aspartic acid peptide or both, thereby inhibiting the progression of neovascularization.
Transport and drug efficacy studies of micro- and nanoparticles administered via periocular injection have been published. Amrite and Kompella determined that subconjunctivally administered nanoparticles and microparticles, of 200 nm and larger, could be retained at the injection site in rats for at least 2 months [106
]. Amrite et al.
showed that periocular blood and lymphatic circulation affected the clearance rate of 20-nm particles administered through periocular injection in dead and living rats, observing only minor transport across the sclera and insignificant transport across the sclera–choroid–RPE [107
]. Chiang et al.
observed in vivo
sustained-release kinetics for more than 1 week using a subconjunctival injection of PLGA microspheres loaded with 5-FU injected in rabbits [108
]. In an effort to develop a sustained delivery treatment for choroidal neovascularization, Saishin et al.
prepared PLGA glucose microspheres incorporating PKC412, a kinase inhibitor that blocks receptors for VEGF, thereby inhibiting ocular neovascularization [109
]. The PKC412-loaded microspheres were administered by periocular injection in young pigs after rupture of Bruch’s membrane via laser photocoagulation. Drug levels were detectable in the choroid, vitreous and retina 20 days postinjection. Ayalasomayajula and Kompella showed that celecoxib, a selective COX2 inhibitor, given orally could inhibit VEGF in a streptozotocin-induced diabetic rat model [110
] and retinal celecoxib delivery improved via a subconjunctival administration [111
]. In an effort to sustain retinal celecoxib delivery, Ayalasomayajula and Kompella then incorporated celecoxib into PLGA (85:15) microparticles and administered them subconjunctivally in rats [112
]. Retinal drug levels were maintained for a 2-week period and inhibited diabetes-induced retinal oxidative stress. Amrite et al.
were able to inhibit diabetes-induced elevations in prostaglandin E2, VEGF and blood–retinal barrier leakage using a posterior subconjunctival (periocular) injection of celecoxib-loaded PLGA microparticles in a streptozotocin diabetic rat model [113
]. Therapeutic concentrations of celecoxib were maintained in the retina in vivo
for 60 days and resulted in no damage to the retina or periocular tissues.
In summary, nano- and microparticles have shown great potential for expanding the arsenal of drug-delivery systems available for treating posterior segment disease due to their ability to provide sustained delivery and reduce complications that result from treatments requiring multiple injections. iv. administration of nanoparticles with surface modifications that can target the retina was a novel approach demonstrated in studies by Sakai et al.
] and Singh et al.
]. Transscleral delivery of anti-VEGF drugs loaded in PLA or PLGA nano- and microparticles is gaining much attention as a feasible and effective method of administration for the treatment of posterior segment disease [12
]. Herrero-Vanrell et al.
] and Barcia et al.
] proved the feasibility of particle sterilization using γ-radiation at an effective USP sterilizing dose of 25 KGy (2.5 Mrad) [114
], which will advance efforts toward clinical trials.