PLA and PLGA microparticles for intravitreal drug delivery: an overview

J. DRUG DEL. SCI. TECH., 17 (1) 11-17 2007

PLA and PLGA microparticles for intravitreal drug delivery: an overview R. Herrero-Vanrell*, I.T. Molina-Martinez Pharmacy and Pharmaceutical Technology, School of Pharmacy, Avda. Complutense s/n, Complutense University, 28040 Madrid, Spain *Correspondence: [email protected] Disorders affecting the posterior segment of the eye are one of the major causes of blindness in western countries. Treatments of these pathologies often require multiple intravitreal injections to achieve effective concentrations in the vitreous cavity. However, repeated injections are poorly tolerated and are frequently associated with cataract formation, retinal detachment, and endophthalmitis. Furthermore, the risk of adverse effects increases with the frequency of intravitreal administration. Injectable microparticles offer a good alternative to multiple injections because they can be administered as conventional intraocular formulations. Among them, microspheres prepared from biodegradable polymers such as poly(lactic) (PLA) acid and poly(lactic-co-glycolic)acid (PLGA) have the advantage of disappearing from the site of administration once the active substance has been delivered. This work describes relevant in vitro and in vivo parameters and variables related to the intravitreal administration of PLA and PLGA microspheres for the treatment of diseases affecting the posterior segment. Key words: Intraocular – Drug delivery – Controlled release – Intravitreal – Microspheres – Poly(lactic)acid (PLA) – Poly(lactic-co-glycolic) acid (PLGA).

periocular route (subconjuctival, sub-Tenon and peribulbar injections) where the drug must cross through the sclera to reach the vitreous. In most cases, the treatment of pathologies that affect the posterior segment requires direct intravitreal administration. Intravitreal drug injection is the common route for treating posterior segment disorders. The main advantage of this kind of administration is that the drug is deposited at the target site, which minimizes systemic side effects. Currently, some acute and chronic posterior segment diseases such as proliferative vitreoretinopathy, age-related macular degeneration, recurrent uveitis, diabetic macular edema, endophthalmitis , acute retinal necrosis, and cytomegalovirus retinitis are often treated with repeated intravitreal injections [1-3]. However, multiple intravitreal injections are poorly tolerated and they are frequently associated with cataracts formation, hemorrhages, retinal detachment, and endophthalmitis [2]. The risk of adverse effects increases with the frequency of injections [3]. Furthermore, the low therapeutic index of the majority of drugs used to treat posterior segment diseases may require doses of the active substance to be injected at or near a toxic level for the retina. Sustained drug delivery systems, such as microparticles, offer a good alternative to multiple intraocular injections. These systems have been under evaluation for intravitreal drug delivery purposes for the past twenty years [4-9]. The devices employed for intravitreal administration are made from either biostable (non-biodegradable) or erodible (biodegradable) materials [10-13]. Biodegradable devices have an advantage over non-biodegradable devices because they disappear from the site of action after delivering the drug. Based on their size, the systems are implanted through a scleral incision [14], thin tissue perforation [1517] or fine needle, as in the case of microparticles [3]. Currently, there are several intraocular devices for drug delivery to the posterior segment that are under clinical use or investigation (Vitrasert, Retisert, Medidur, Psorudex and I-Vation) [18]. However, most of these devices are not biodegradable, and surgery is required for their removal or reimplantation. Injectable particles and colloidal systems such as micro(nano)spheres, micro(nano)-capsules, microemulsions, liposomes, and micel-

The eye is divided into two parts: the anterior and the posterior segment (Figure 1). The anterior segment includes the cornea, iris, anterior chamber, crystalline lens, and ciliary body. The posterior segment includes the choroids, retina, retinal pigment epithelium, and vitreous body. Vitreoretinal pathologies are one of the major causes of blindness, mainly because the adjacent tissues can not be recovered once they are damaged. Disorders affecting the posterior segment of the eye can be treated through several routes of administration: topical, systemic, periocular, and intraocular. Although topical administration of active substances is widely used in treating ocular disorders, only a low percentage (approximately 5%) of a topically administered drug is able to reach intraocular tissues [1]. Effective drug concentrations in the anterior segment can only be achieved when large concentrations of drugs are administered. Intravitreal therapeutic concentrations are difficult to attain with systemic administration because of blood aqueous and blood retinal barriers, which limit the distribution of active substances in the eye. One method that has been considered for the treatment of posterior segment diseases is administration through the Anterior segment

Posterior segment

Vitreous Conjunctiva

Cornea

Optic nerve

Aqueous humor

Lens Anterior chamber

Posterior chamber

Retina Sclera

Choroids

Figure 1 - Anatomical features of the eye.

11

J. DRUG DEL. SCI. TECH., 17 (1) 11-17 2007

PLA and PLGA microparticles for intravitreal drug delivery: an overview R. Herrero-Vanrell, I.T. Molina-Martinez

les offer an excellent alternative to implants and multiple intravitreal injections. The main advantage of these systems is that they can be injected as conventional intraocular solutions [3]. In the case of microparticles, higher amounts of drugs can be incorporated because the release time of the active substance is longer. It is important to note that in order to successfully treat vitreoretinal diseases, intravitreal drug concentrations must be maintained for long periods of time. Among the polymers that have been studied for use in preparing particles for intraocular drug delivery are gelatin, albumin, polyorthoesters, polyanhidrides and polyesters, particularly polymers of D,L-lactic acid (PLA) and glycolic acid (PGA), as well as their copolymers polylactic-co-glycolic acid (PLGA). These biomaterials have been used to make microspheres for intravitreal drug delivery. These polymers have demonstrated good biocompatibility and their degradation products (lactic and glycolic acids) [11, 12] are metabolized further into carbon dioxide and water [19]. Thus, PLA and PLGA microspheres are able to release drugs in the vitreous and maintain concentrations of the active substance at a therapeutic level for a long time. Finally, these microparticles disappear from the site of action after releasing the active substance without damaging intraocular tissues. This work gives an overview of relevant parameters associated with the usefulness of PLA and PLGA microspheres for the treatment of diseases affecting the posterior segment.

Figure 2 - (Left) SEM photographs of sodium ganciclovir-loaded microspheres (300-500 µm) prepared from PLGA50:50 (Mw 34,000 Da) and vitamin E as additive. Magnification 100x. (Middle) View of a fragmented microsphere. (Right) Magnification 50x.

I. GENERAL ASPECTS OF MICROPARTICLES 1. Preparation and characterization of microspheres

Several techniques have been described for preparing microparticles such as denaturation or cross-linking of macromolecules in emulsions, aerosol formation, desolvation by solvent evaporation or spray drying, interfacial polymerization, aggregation by pH adjustment or heat, and solvent evaporation from an emulsion (o/w, w/o and w/o/w [2022]). Once prepared, microparticles are characterized morphologically with different techniques, of which light microscopy and scanning electron microscopy (SEM) are the most common. For example, Figure 2 shows micrographs of monolithic microparticles prepared from a PLGA 50:50 (Mw 39,000 Da). The size of microspheres for intravitreal administration has to be adequate for their injection through a suitable needle. For this purpose, granulometric analysis is conducted to calculate statistic diameters and size distribution. Moreover, it is important to ensure that these microparticles are free of any residual traces of organic solvent. In this case, differential scanning calorimetry (DSC) is used to provide this information through thermal profile changes observed for the polymer. This technique can also be applied to physical mixtures of the drug and non-loaded microspheres to detect possible changes in the microparticle components. As an example, Figure 3 shows DSC thermograms of acyclovir, a physical mixture of acyclovir and nonloaded microspheres, and acyclovir-loaded microspheres for intravitreal delivery in which an interaction between the formulation components was observed.

Figure 3 - DSC thermograms of acyclovir (I), acyclovir and non-loaded microspheres (physical mixture) (II) and acyclovir-loaded microspheres (from [28] with permission).

is best to use biodegradable substances because they remain in the microspheres after their maturation. Herrero-Vanrell et al. developed PLGA (Mw 34,000 Da) microspheres for intraocular administration that were loaded with sodium ganciclovir in which the addition of a non-biodegradable substance (fluorosilicone oil) to the inner phase of the emulsion modulated the release rate of the hydrophilic drug. Although the additive used did not degrade into the vitreous, the authors stated that the residual ocular oil should not present an intolerance problem [24]. In order to prevent residual components in the vitreous cavity after injecting the microspheres, the same authors developed sodium ganciclovir microspheres with biodegradable vitamin E (α-tocopherol) and mygliol as additives [25-27]. In those experiments, the in vitro release rate of sodium ganciclovir fit into a zero-order kinetic model and presented lower initial bursts than the rates observed without additives (Figure 4). When microspheres are intended for the administration of drug in a relatively isolated area such as vitreous, the active substance must be released from the microparticles so that therapeutic levels can be attained with the minimum dose. To reach this objective, MartinezSancho et al. found that adding gelatin to the external phase of an o/w emulsion increased the release rate of acyclovir, while adding isopropyl-miristate decreased the release rate of the drug [28]. These authors reported a 40% reduction in the dose of acyclovir microspheres to be administered when gelatin was added to the external phase of the emulsion, compared to microparticles without additives [28]. In these experiments the polymer used was PLGA50:50 (Resomer RG 502, Mw 15,000 Da) and the size of the microspheres was 46 µm.

2. Drug release rate from microparticles and additives

Generally, the drug release rate of microparticles is affected by polymer molecular weight and composition, drug loading, active substance solubility, and total surface area of the particles. Usually, small microspheres have lower amounts of encapsulated drug and release the active substance faster than higher ones (23). The in vitro release profile can be controlled by modifying the components of microparticles. The release rate of drugs from microspheres can be modulated to some extent by adding several substances. If the solvent evaporation from an emulsion technique is being used to prepare the microspheres then additives can be added to the inner or external phase of the emulsion. If adding to the inner phase, it 12

PLA and PLGA microparticles for intravitreal drug delivery: an overview R. Herrero-Vanrell, I.T. Molina-Martinez

J. DRUG DEL. SCI. TECH., 17 (1) 11-17 2007

practices [43]. This dose, defined by the USP as an effective sterilizing dose, has been used for the sterilization of polymeric medical devices. Nevertheless, gamma irradiation can alter PLGA structures, reducing the Tg values of polymers [35]. In this case, the relatively low glass-transition temperatures could favor subsequent reactions of free radicals due to the high mobility of the polymeric chains. PLA and PLGA microspheres have proven to be sensitive to γ-irradiation. It has been described that γ-irradiation of microspheres made of low and high molecular weight PLGA [45] at room temperature lowered Mw and Mn. Furthermore, gamma irradiation is associated with temperature increase [46], which can cause microsphere fusion during irradiation exposure. In some cases, the process causes the in vitro drug release from biodegradable microparticles to increase [47]. Taking into account that terminal sterilization is preferred to aseptic processing for any drug or device before it is placed in the eye, Herrero-Vanrell et al. evaluated the effect of final sterilization on the properties of ganciclovir-loaded PLGA (inherent viscosity 0.2 dl/g) microspheres [23]. In the reported experiments, microspheres were sterilized by gamma radiation (25 kGy), with care taken to avoid a temperature increase during irradiation. The authors concluded that this irradiation dose, under the conditions of this experiment, produced no significant changes in the release rate of the drug from the microspheres. Similar results were reported by Fernandez-Carballido et al. and Martinez-Sancho et al. for indomethacin and acyclovir PLGA microspheres, respectively. The processes were carried without a temperature increase during irradiation. In all cases, a similarity between release curves before and after sterilization was demonstrated [43, 48]. The similarity factor (f2) allows characterizing the release profile of the active substance before and after microparticle sterilization. Conceptually, f2 is a measurement of the similarity in the percentage of dissolution between two curves. The values of f2 range from 0 to 100 with a higher similarity factor value indicating higher similarity between the two release profiles [49, 50]. The similarity limits for average difference distances is shown in Table I.

120

Released ganciclovir (%)

100

80

60

Without additive

40

alpha-tocopherol 20

Miglyol

0 0

5

10

15

20

25

30

35

40

45

Time (days) Figure 4 - Ganciclovir cumulative release profiles in 1.5 ml PBS from 10 mg of PLGA (50:50) microspheres with biodegradable additives and additive-free microparticles (from [25] with permission).

A significant challenge in intravitreal drug delivery is the use of substances with therapeutic activity as additives. One example of this is retinoic acid (vitamin A), which has antiproliferative properties that can eliminate the inconvenience of successive intraocular injections [30]. Retinoic acid was added to the acyclovir PLGA microspheres resulting in an adequate, long-term release of both drugs. Furthermore, the additive improved the release rate of acyclovir [31]. In this formulation vitamin A modifies the release rate of acyclovir from the microspheres and potentially prevents the adverse effects of intravitreal injections.

3. Injectability of microparticles

Microparticle size widely determines drug release [32, 33] and can significantly limit intravitreal administration. A suitable diameter for intraocular administration has been previously described [33, 34]. For clinical purposes, microspheres should pass through 18-30 needles. In general, the size of microspheres being used for intravitreal administration is lower than 150 µm in diameter [35]. Microsphere suspensions have been injected with different diameter needles such as 27-gauge (G) [36-38], 26-gauge [39, 40], and 18-gauge needles [41], as well as a microinjector connected to a glass micropipette (tip diameter 40 µm) [42]. Microparticles can be administered through a needle properly. For this reason, injectability of microparticles is one of the critical parameters in clinical practice. Microsphere injectability depends on particle size, as well as the syringe, needle size, and properties of the solvent used for microparticle suspension. Applying a maximum ejection force of 12 N over 10 s can be considered in practical experience as an acceptable development criterion for defining the target for a minimum inner diameter of a needle [43]. Based on this assumption, injectability assays are conducted by registering the maximum force needed for injecting the microsphere suspension.

Table I - Average differences between dissolution profiles of two batches expressed in percentages (%) with the corresponding similarity factor values (f2). Average differences

2%

5%

10%

15%

20%

f2 limits

83

65

50

41

36

Two curves are considered to be similar when the f2 values are equal or higher to 50 (lesser than 10% of difference). An example of two similar release profiles obtained before and after γ-irradiation of ganciclovir PLGA microspheres is shown in Figure 5.

5. Determination of the theoretical amount of microparticles for intravitreal injection

Local toxicity is based on the amount of microspheres used; it is important to note that when microspheres are prepared for administration in a relatively isolated zone such as the vitreous cavity the therapeutic drug levels can be attained with the minimum amount of particles. The dose of microspheres to be injected into the vitreous can be theoretically calculated from the following equations:

4. Sterilization of microspheres

Sterility is extremely important for intraocular systems such as microparticles. Ethylene oxide sterilization is not recommended because of its carcinogenic properties and undesirable residues in the microparticles, yet ultraviolet radiation does not ensure total sterility. A gamma-irradiation dose of 25 KGy (2.5 Mrad) is generally accepted for sterilizing pharmaceutical products without providing any biological validation, and it is in accordance with good manufacturing

K0 = Css x Cl

Eq. 1

K0 = Css x Ke x Vd

Eq. 2

where K0 is the in vitro release rate that can provide therapeutic levels in the vitreous, Css is the steady-state concentration that needs to be attained and maintained in the vitreous, Cl is the drug clearance from 13

J. DRUG DEL. SCI. TECH., 17 (1) 11-17 2007

PLA and PLGA microparticles for intravitreal drug delivery: an overview R. Herrero-Vanrell, I.T. Molina-Martinez

of dexametasone PLGA50:50 (Resomer RG 502) microspheres were injected into rabbits. Microparticles agglomerated and remained up to 45 days after their administration [53]. In both cases, the authors reported that, histologically, there was no retinal or choroidal damage caused by the injection of microparticles. Generally, the reaction described after intraocular injection of microparticles is similar to that described for microspheres injected intramuscularly into rabbits in which biodegradation was almost completed by day 63 [54]. The foreign body reaction gradually decreased with time, and according to several authors, at twelve weeks after administration, there were only fragments of microparticles in the site of implantation [37, 41]. Histopathological studies have been conducted to study the inflammatory reaction to microspheres injected into the vitreous cavity. Veloso et al. [41] described a mild localized foreign body reaction after the injection of 10 mg of ganciclovir PLGA (inherent viscosity 0.39 dl/g) microparticles. Although hystopathological analysis at four and eight weeks after administration revealed the existence of mononuclear cells and multinucleated giant cells, there was no damage to the retina or other adjacent structures. Moreover, any inflammatory reaction in the vitreous or localized foreign body reaction due to microsphere injection decreased with time. It should be noted that one special aspect of microparticles being used for intraocular administration is that they should not interfere with the visual pathway. Care should be taken when injecting the microspheres to avoid interfering with the visual pathways (microparticles are not transparent) [55].

110

Cumulative released Ganc.(%)

100 90 80 70 60 50 40 30 20 10 0 0

4

8

12

16

20

24

28

32

36

40

44

Time (days)

Figure 5 - Percentage of ganciclovir released in PBS (1.5 ml) from 10 mg of PLGA (50:50) microspheres before (O) and after (Δ) their exposure to an effective dose (25kGy) of γ-radiation. Microspheres size 300-500 µm (from [23] with permission).

the vitreous, Ke is the constant elimination rate, and Vd the vitreous volume (1.5 ml in rabbit eyes and 4.5 ml in human eyes) [23]. The amount of microspheres can be calculated from the value of the drug release rate from microparticles (µg/day per mg of microspheres) obtained from the release studies.

4. Intravitreal degradation of microparticles

II. INTRAVITREAL ADMINISTRATION OF MICROPARTICLES 1. Preparation of microsphere suspensions

The site for microparticle injection is the pars plana, at 3 to 4 mm from the limbus. At this site, a scleral puncture is made through which the needle is introduced into the vitreous cavity, with the bevel of the needle positioned upward [3, 27, 41]. Then, microparticles are injected into the vitreous at an appropriate angle and depth to reach the midvitreous, thus avoiding lens damage. Administration is carried out slowly, and before removing the needle, an occlusion of the scleral hole is maintained for 1 min to avoid reflux. In several cases, a vitrectomy is performed before the microspheres are injected [3, 40, 41]. This surgical procedure is performed before the microsphere injection.

Generally, microspheres are described as degrading faster in vivo than in vitro. It is important to make sure that the microspheres are completely eliminated from the vitreous after their administration. After intravitreal injection of 5-fluorouracil (5-FU), its in vitro release from PLA (3,400 and 4,700 daltons) and PLGA 70:30 (3,300 daltons) microspheres (diameter 50 µm) was evaluated by Moritera et al. [40]. The 5FU was released over two to seven days in vitro, with faster release for the PLGA polymer. Approximately 70% of 5FU was released from the 4,700-daltons PLA microspheres within seven days. In the in vivo studies, 85% of the encapsulated 5FU was released from the PLA 3,300-daltons microspheres within seven days, and 98% of the drug was released from the PLGA microspheres in just two days. The authors also reported that the drug was released faster from the injected microspheres in pathologic rabbit vitreous than normal rabbit vitreous. Microspheres gradually become smaller and finally disappeared from the normal rabbit vitreous in 48 days. However, clearance from the vitreous cavity was two weeks faster in eyes that had undergone a vitrectomy: when the polymerʼs molecular weight increases, the residence time of microspheres in the vitreous cavity increases. Veloso et al. [41] described the presence of microparticles in rabbit eyes up to eight weeks after the injection of 5, 10 and 15 mg of ganciclovir PLGA (50:50, Mw = 34,000) microspheres. In turn, Giordano et al. evaluated the intravitreal biodegradation and clearance time of PLGA microspheres prepared with a low molecular weight PLGA polymer (50:50 Resomer 502) without active substance in vitrectomized rabbits. In this study, they observed evidence of microspheres up to 24 weeks after injection [37]. These results suggest that the presence of the drug, as reported by other authors, may affect the degradation time of microparticles.

3. Intraocular tolerance to microparticles

III. TARGET VITREORETINAL DISEASES

Microparticles are administered into the vitreous cavity as easily as a conventional injection. First, microparticles are suspended in a vehicle. The most frequently-used solvents for administering particles are phosphate buffer solutions (PBS) or balanced salt solutions (BSS) with a pH of 7.4 [3]. Because suspended microparticles tend to stick in the syringe, some researchers have described the employment of viscous vehicles [27, 41] for better intravitreal administration. The viscous solutions used to suspend the particles are physiological solutions of hyaluronic acid or hydroxypropylmethylcellulose, which are commonly used as vitreous substitutes in ophthalmology [51, 52]. Both polymeric solutions are transparent, biocompatible, and dilute quickly in the intraocular fluids before being finally eliminated from the eye.

2. Injection of microspheres

Biodegradable microparticles may be a potential option in the treatment of vitreoretinal diseases that require intravitreal injections. Currently, posterior segment diseases such as proliferative vitreoretinopathy, age-related macular degeneration, diabetic macular retinopathy, recurrent uveitis, macular edema, endophthalmitis, acute

Different reactions to microparticles after their injection have been described. Khoobehi et al. observed a whitish material in the vitreous cavity for ten days after the injection of fluorescein-loaded microspheres, which then disappeared after 20 to 25 days [36]. Herrero-Vanrell et al. described a similar observation after 10 mg 14

PLA and PLGA microparticles for intravitreal drug delivery: an overview R. Herrero-Vanrell, I.T. Molina-Martinez

J. DRUG DEL. SCI. TECH., 17 (1) 11-17 2007

retinal necrosis, and cytomegalovirus retinitis are often treated with repeated intravitreal injections [1-3]. Proliferative retinopathy (PVR) is one of the major causes of blindness following surgery for retinal reattachment [56]. The treatment of PVR includes the use of agents with antiproliferative activity. Microspheres loaded with active substances such as adriamycin [39], 5 FU [40, 57] and retinoic acid (vitamin A) [58] have been evaluated for the treatment of PVR in animal models. In all reported studies, the rate or incidence of retinal traction detachment decreased after injection of microspheres. Age-related macular degeneration (AMD) is the most common cause of blindness in the elderly populations of western countries. The exudative form of AMD might lead to choroidal neovascularization (CNV). PLGA microspheres loaded with anti-endothelial growth factor (VEGF) have been suggested for reducing the formation of new blood vessels in the eye [59, 60]. The term “uveitis” is used to denote any intraocular inflammatory condition without reference to the underlying cause [61]. In fact, uveitis is considered to be an intraocular autoimmune or inflammatory disease involving the ciliary body, choroids and/or adjacent tissues. The disease has both acute and chronic manifestations. Current treatment for chronic features usually includes topical, periocular, or systemic corticosteroids [62, 63]. Transitory therapeutic drug levels can be attained through the administration of steroids by intravitreal injections. Nevertheless, therapeutic drug concentrations are difficult to attain in the vitreous for a prolonged period of time due to the short, intravitreal half-life of corticosteroids [64]. For these reasons, repeated intravitreal injections are needed to maintain therapeutic levels for a long period of time. Dexamethasone microspheres prepared from poly (D,L-lactide-co-glycolide) (50:50) with an inherent viscosity of 0.2 dl/g were developed to provide sustained release of the drug for the treatment of uveitis. Ten milligrams of microspheres (1410 µg of dexamethasone) released the drug in vitro for approximately 45 days [65, 66]. Macular edema is usually treated with corticosteroids, among which triamcinolone acetonide is the most commonly used. Recently, Cardillo et al. [66] have presented the first study in which the administration of PLGA microspheres loaded with triamcinolone have been injected into the human vitreous. The authors reported preliminary results with good tolerance for the PLGA microparticles. There has been an increase in fungal endophthalmitis recently due to the higher immunosuppression associated with organ transplantation and malignancies, as well as the increased use of antibiotics and immunosuppressive agents. Biodegradable microparticles loaded with antibiotics could deliver effective concentrations of the active substance into the vitreous cavity. Acute retinal necrosis (ARN) is a viral infection characterized by necrosis of retinal cells that can lead to irreversible blindness. Some herpes viruses that infect humans are herpes simplex virus (HSV) types 1 and 2, varicella zoster, and Epstein-Barr viruses. The therapy for ARN usually involves intravenous or intravitreal administration of acycloguanosine (acyclovir). Intravitreal administration of acyclovir has demonstrated to be more effective than the intravenous administration of the drug and have fewer side effects. Although the intravitreal therapy is effective, the relatively high dose that is required has untoward side effects. Conte et al developed a controlled release formulation from different PLA and PLGA polymers loaded with acyclovir using the spray drying technique. In vivo evaluation was performed by injecting 0.5 mg of microparticles (25 µm diameter) from D,L-PLA (28,000 Da) into rabbit eyes. Drug levels were detected in the vitreous for fourteen days after the microspheres were administered [38]. Choudhury and Mitra have described guanosine-loaded PLGA (75,000 to 100,000 daltons) microspheres developed for a drug release of one week after intravitreal injection of the microspheres [67]. Cytomegalovirus (CMV) retinitis occurs in acquired immunode-

ficiency syndrome (AIDS) and other immunosuppressed conditions. The CMV infection is progressive and can result in blindness from retinal detachment associated with retinal necrosis [68, 69]. Although intravitreal ganciclovir injections provide effective intraocular drug concentrations, frequent injections are required to maintain therapeutic drug levels [70]. Veloso et al. tested the antiviral effect of ganciclovir released from PLGA microspheres in rabbit eyes inoculated with the human cytomegalovirus (HCMV). One 10-mg injection of 300-500 µm ganciclovir-loaded microspheres prepared from PLGA 50:50 (inherent viscosity 0.39 dL/g), containing 864.04 µg of the drug controlled the progression of fundus disease in the HCMV-inoculated rabbit eyes [41]. Neuroprotection has been proposed as a therapeutic option for the treatment of glaucoma. This treatment focuses on promoting the survival of retinal ganglion cells (RCG). RCG survival can be achieved by neurotrophins. PLGA50:50 (Mw 25,000 Da) microspheres containing Glial-cell-line-derived neurothropic factor (GDNF) were assayed in mice. In the reported work microspheres loaded with GDNF increased long-term RGC survival in a spontaneous glaucoma model [70].

IV. FUTURE STUDIES

Currently, there is a significant amount of experimental evidence indicating that intravitreal administration of PLA and PLGA microparticles loaded with active substances can provide therapeutic drug levels to the vitreous cavity. This approach to intraocular drug delivery shows great promise in the treatment of devastating eye diseases because the systems are biodegradable, and they degrade into metabolic products that are non-toxic to both the retina and its surrounding tissues. Moreover, microspheres can be injected through a needle instead of a surgical procedure. Microparticles are able to incorporate higher amounts of drugs than other systems, providing long term release of the active substance. Special care is needed to avoid the risk of microsphere sedimentation in the vitreous after microspheres injection. Current and future studies will be able to further explain the usefulness of this approach.

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ACKNOWLEDGEMENTS Research support: Complutense Research Groups (UCM2005-920415) and SAF(2004-06119-C02).Acknowledgements are given to copyright holders for their permission to reprint figures.

MANUSCRIPT Received 3 July 2006, accepted for publication 20 December 2006.

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