Current Drug Delivery

Current Drug Delivery

930

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Current Drug Delivery, 2018, 15, 930-940

REVIEW ARTICLE ISSN: 1567-2018 eISSN: 1875-5704

Impact Factor: 2.078

Biodegradable Microspheres as Intravitreal Delivery Systems for Prolonged Drug Release. What is their Eminence in the Nanoparticle Era?

Pore Pore

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Nerve Nerve Fatlobules lobules Fat

Elisabetta Gavini1, Maria C. Bonferoni2*, Giovanna Rassu2, Antonella Obinu3, Franca Ferrari2 and Paolo Giunchedi1 1

Department of Chemistry and Pharmacy, University of Sassari, Sassari, Italy; 2Department of Drug Sciences, University of Pavia, Pavia, Italy; 3Experimental Medicine, Department of Clinical-Surgical, Diagnostic and Paediatric Sciences, University of Pavia, Pavia, Italy

Received: October 28, 2017 Revised: December 16, 2017 Accepted: February 14, 2018 DOI: 10.2174/1567201815666180226121020

Abstract: Drug administration to the posterior segment of the eye has many challenges due to the natural barriers and consequent problems of low and unpredictable bioavailability. There is an increasing need for managing severe posterior eye diseases, such as age-related macular degeneration, diabetic retinopathy, etc. Most of these diseases, if left untreated, lead to blindness. Traditional ocular formulations and topical administrations are almost inefficient and the drug delivery to the back of the eye requires direct administrations through intravitreal injections of innovative drug delivery systems. These systems must be easily injectable, able to release the drug for a prolonged period of time (to overcome the problem of repeated administrations) and made of biodegradable/biocompatible polymers. Among these delivery systems, microspheres still have an important role. This overview wants to highlight the use of microspheres as intravitreal systems to overcome the challenges of back of the eye diseases. Studies have shown that microspheres are able to enhance the intravitreal half-life and thus bioavailability of many drugs, protecting them from degradation. Furthermore, personalized therapies can be made by changing the amount of administered microspheres. This review focuses on the materials (polymers) used for the preparation of the microparticulate systems and comparative remarks are made with respect to the use of nanoparticles.

Keywords: Posterior segment, intravitreal injections, repeated administrations, prolonged release, microspheres, nanoparticles, biodegradable polymers. 1. INTRODUCTION The eye globe can be anatomically divided into anterior and posterior segments, which correspond to about one-third and two-thirds of ocular region, respectively [1]. The posterior segment presents three layers (sclera, choroid and retina) that surround the vitreous body, an internal cavity (volume about 4 mL) filled with an internal gel structure mainly constituted of water (about 99%) and containing collagens, noncollagenous proteins, glycosaminoglycans, hyaluronic acid and proteoglycans [2]. The internal structure of the eye globe depends on the properties of these substances. The posterior eye segment can be affected by severe pathologies such as age-related macular degeneration (AMD), diabetic macular edema (DME), diabetic retinopathy (DR), viral retinitis, retinal vein occlusion (RVO), choroid neovascularization (CNV) and posterior uveitis [3]. Generally, back *Address correspondence to this author at the Department of Drug Sciences, University of Pavia, viale Taramelli 12, 27100 Pavia, Italy; Tel: +39-0382987375; E-mail: [email protected] 1875-5704/18 $58.00+.00

of the eye diseases are chronic and degenerative, some of them are related to the elderly and they can lead to vision loss. These diseases are increasing in the aging populations and nowadays it is calculated that millions of people are affected worldwide. The therapies for these diseases are therefore crucial because their success means the maintenance of a good quality of life for the patients. However, the therapies for the posterior segment of the eye are problematic for different reasons. They must be initiated at early stages of the pathology; the drug delivery to the posterior ocular segment is significantly impaired for the anatomical barriers and the physiological characteristics of the district, with consequent problems of low and unpredictable bioavailability; furthermore, these diseases are often chronic. Therefore, to be sure of the success of the therapies, repeated administrations of the active agents into the posterior part of the eye are required and these administrations must be of therapeutic concentrations and for prolonged periods of time in correspondence with the target sites. © 2018 Bentham Science Publishers

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INCREASE IN IN PATIENT PATIENT COMPLIANCE COMPLIANCE INCREASE

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Biodegradable Microspheres as Intravitreal Delivery Systems

The possible routes that permit to achieve, directly or indirectly and with different degrees of efficiency, the posterior part of the eye can be classified into topical, systemic, periocular and intravitreal [4]. The topical drug administration represents an indirect way to reach this target. It is characterized by high patient compliance and it has the possibility of self-administration. It is the preferred route to treat the anterior segment of the eye or the ocular surface, but unfortunately, it is not efficient in obtaining therapeutic drug concentrations to the posterior segment owing to the low permeability of the corneal epithelium and the rapid clearance from the surface of the eye [5]. The poor bioavailability of drugs topically administered limits, therefore, their access to the intraocular tissues. The systemic drug absorption is not the optimal choice owing to the blood-retinal barrier (BRB): systemic administrations with the goal of posterior eye segment require high drug doses to achieve adequate therapeutic levels with the consequent high risk of possible adverse effects [5]. Topical and systemic administrations are “non-invasive” because they do not involve either injections or scleral incisions / perforations. The periocular route involves the region surrounding the eye and permits the drug delivery in correspondence of the external surface of the sclera. In case injections, incisions or perforations are involved, this route is an “invasive” method of drug administration; for example, microneedles having a length of about 800 m, have been used in rabbits for the injection of bevacizumab into the suprachoroidal space [6]. It is more efficient with respect to topical and systemic administrations [4], in general, it can be considered less invasive with respect to intraocular administrations, more than topical and systemic routes. Intravitreal (IVT) injection of drugs to the eye involves the direct injection of the drug-loaded formulation, as solutions, suspensions containing (micro/nano)particles, depots or implants, into the posterior segment, bypassing the bloodocular barrier (Fig. 1).

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intraocular bioavailability by overcoming all the barriers of the posterior eye segment through a direct administration. However, IVT injections present some problems. They are “invasive” because partially break the ocular tissue integrity; they are characterized by low patient compliance; there is no possibility of self-administration; the IVT injections are done by health care providers. Furthermore, there is the problem of drug clearance from the vitreous. The elimination rate is strongly influenced by the rate of drug diffusion through the vitreous; the drug is eliminated from the vitreous humour via the anterior chamber and across the retinal surface [9]. The process of elimination generally occurs with a first-order rate [10]. If the drug is administered as a solution, the clearance from the vitreous is usually a fast process, however, in several cases drug elimination occurs in a few hours and therefore repeated IVT injections are required. Larger molecules are retained in the vitreous for longer periods (days), while molecules that have a Mw lower than 500 Da are rapidly cleared. Consequently, frequent administrations are required owing to the limited retention half-life. The process of elimination is further complicated in elderly patients with collapsed vitreous structure [9]. In relationship with the frequency of the injections, possible serious disadvantages such as hemorrhage, retinal detachment, cataract formation, increase in intraocular pressure, degeneration of photoreceptors and endophthalmitis can occur [11]. An important example of these problematics is the administration of antivascular endothelial growth factors (antiVEGFs). Food and Drug Administration (FDA) has approved therapies based on anti-VEGFs for choroidal neovascularization (CNV). Anti-VEGFs have shown remarkable results for the treatment of posterior ocular diseases, but they need frequent IVT injections [12]. These drugs, in fact, are rapidly cleared from the vitreous cavity [13]. The repeated administrations of anti-VEGFs are characterized by side effects such as elevation of intraocular pressure (IOP), retinal detachment, retinal hemorrhage and endophthalmitis [14, 15]. To achieve less-frequent administrations, controlled/prolonged drug release formulations are strongly needed to improve patient’s compliance and lower side effects associated with frequent injections. Different IVT controlled delivery systems able to release the drug for a prolonged period of time have been developed to overcome such problems: for example, microspheres, nanoparticles, liposomes, niosomes and more recently dendrimers [16].

Fig. (1). Intravitreal injection of drug formulations: a way to reach directly the posterior part of the eye.

Nowadays IVT drug delivery can be considered the most efficient route for posterior segment [7, 8]. It provides high

In the area of intravitreal dosage forms, as in other areas of drug formulations, there is an impressive (and increasing) number of papers that describe formulations based on nanoparticles. However, this is a case in which many examples of recent papers describing microsized formulations are still present. Does this still have a rational? The aim of this review is to define the present situation regarding the use of microspheres as controlled release systems for the intravitreal administration of drugs and to understand if there is still a rationale for the preparation of microspheres in the “nanoparticle era”. The various drug-delivery strategies used for IVT therapies to achieve a sustained drug release involving microspheres and putting into evidence the good results

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and performances that can be obtained using these microsized systems will be discussed. 2. PARTICLE SIZE AND RESIDENCE TIME IN THE VITREOUS BODY As reported in the literature, both nanosized and microsized particles are used for intravitreal administrations, because particulate (micro and nano) carrier systems enhance intravitreal drug residence as they are little affected by vitreous clearance mechanisms [17] and therefore they are used as prolonged drug release formulations. However, a direct correlation between the size of the carrier and its residence time and behavior in the vitreous environment is not always clear. Sakurai et al. [18] injected suspensions of polystyrene micro/nanoparticles (2 m, 200 nm and 50 nm in diameter) containing fluorescein (Fluoresbrite® Plain Microspheres, Warrington, Pa., USA) into the vitreous body of unilateral rabbit eyes. A solution of sodium fluorescein (same concentration) was injected as the control into the vitreous cavity of the other eye. Intraocular kinetics of particles was determined using a scanning fluorophotometer with the vitreous fluorescence measure. Within 3 days the fluorescence in the control eyes completely disappeared, while the fluorescence derived from particles was observed in the vitreous cavity for several weeks, but vitreous half-lives were shorter for higher microparticle sizes (2 m, t1/2 = 5.4 + 0.8 days) with respect to nanoparticles (200 nm: t1/2 = 8.6 + 0.7 days, 50 nm: t1/2 = 10.1 + 1.8 days). Furthermore, 1 month after the administration of microspheres (2 m diameter) there was a dense vitreous opacity and deposits of particles were found by indirect ophthalmoscopy on the retina. On the contrary, smaller nanospheres (200 nm diameter) were homogeneously dispersed in the vitreous cavity. This study involved micro/nanoparticles made of a polymer (polystyrene) which is not biodegradable. The use of biodegradable particles is preferable. However, the authors in this study did not use biodegradable particles because they claimed that the diameter of such particles change in the time owing to the erosion due to biodegradation and for this reason, a relationship between particle size and intraocular distribution cannot be determined precisely. To the best of our knowledge, unfortunately, no similar studies have been carried out on biodegradable particles. More recently, Xu et al. used a rheologic technique (realtime multiple particle tracking, MPT) for the study of the movements of polystyrene nanoparticles of various sizes and surface chemistry in fresh bovine vitreous [19]. The vitreous gel is structurally fragile and after removal from the eye, the structure of the vitreous can be disrupted simply by the gravitational force. For this reason, an interesting approach has been developed to access the central part of the vitreous, preserving the vitreous gel in its native state [19]. Polystyrene micro/nanoparticles with different sizes and surface chemistry were gently injected (as suspensions) into the vitreous, using a 30-gauge 10 l Hamilton syringe. The originality of the work was in the use of micro/nanoparticles differing not only for particle size but also for surface characteristics. Nanoparticles (size around 500 nm) rapidly pene-

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trated the vitreous gel when coated with polyethylene glycol (PEG), while microparticles (size higher than 1-2 m) were highly restricted owing to steric obstruction. Polystyrene nanoparticles coated with primary amine groups (-NH2) showed positive charge surfaces at the pH of bovine vitreous (pH about 7.2), and therefore were immobilized within the vitreous gel. On the contrary, polystyrene nanoparticles (size range of 100-200 nm) coated with carboxylic groups ( COOH) and possessing negative charge surfaces readily diffused through the vitreous gel at lowest concentrations (below 0.0025% w/v), while at higher concentrations (about 0.1% w/v) the nanoparticles led to aggregates in the vitreous. This work, therefore, shows as in the case of nanoparticles the surface characteristics and concentrations are very important in determining the behaviour of the particles in the vitreous body while in the case of microparticles the size and the consequence steric interactions in the gel structure are more important. 3. MICROSPHERES FOR INTRAVITREAL ADMINISTRATION Microparticles (size of 1-1000 m) are polymeric carriers. Depending on their structure, they are classified as microcapsules (having a “reservoir” structure) and microspheres (having a “matrix structure”). A further classification can be made depending on the nature of the polymer(s) used for their preparation. Microspheres can be administered through the ocular routes [20-22]. Among them, intravitreal microparticulate systems play an important role. A general view can be seen in Table 1, where microspheres for intravitreal use are classified depending on the polymeric material used for their preparation. For the intravitreal injection, suspensions are prepared by dispersing the microspheres in a liquid injectable medium. Syringeability and injectability are parameters of great importance for intraocular formulations. Microspheres are injected as suspensions so special care must be taken in the preparation of homogeneous particle dispersions in the clinical practice. Isotonic phosphate buffer solutions of pH 7.4 are mainly used as vehicles. Solutions composed of viscosity enhancers can also be employed to delay the clumping of particles. If microspheres clogging occurs, biopolymers such as hydroxypropylmethylcellulose or hyaluronic acid added in the aqueous vehicle help improve both syringeability and injectability. With the intravitreal injection, the formulations are directly placed into the body of the eye. For this reason, the polymers used must be biodegradable/biocompatible: this means they have the important advantage that they gradually disappear from the site of implantation during and/or after the release process. At the end, no further operation is needed to remove the exhausted microspheres because they are disappeared. What makes them attractive to treat eye diseases is their capacity of controlling drug release even for long periods of time, after their direct administration into the ocular target and to biodegrade during and after the drug release process. These microsystems are used to load both lipophilic and hydrophilic drugs.


Table 1.


Polymer-based classification of microsized intravitreal formulations. Polymer (Microspheres)

Drug

References

Poly(lactic acid) / poly(lactic-co-glycolic acid)

Fluorescein sodium (dye)

[26]

Poly(lactic-co-glycolic acid)

Adriamycin

[27]


Acyclovir

[28-31]


Acyclovir and vitamin A palmitate

[32]


Guanosine

[34]


5-fluorouracil

[35]


Dexamethasone

[40]


Dexamethasone and vitamin E

[43]


Triamcinolone acetonide

[45, 46]


Ranibizumab

[47]


4-(3-chloroanilino)-6,7-dimethoxyquinazoline (EGFR TKI)

[48, 49]


Glial cell line-derived neurotrophic factor (GDNF) and vitamin E

[52-54]


Proinsulin / Insulin

[55]

Poly(lactic acid)

TG-0054

[56]

Polyethylene-poly(lactic acid) (PEG-PLA) block-copolymers

Tat-EGFP

[59]

Poly--hydroxybutyrate

Doxorubicin

[62]

Poly(ester amides)

Dexamethasone

[65]

Porous silicon

Daunorubicin

[68]

Porous silicon

Dexamethasone

[69]

Chitosan

Tat-EGFP

[84]

Oligochitosans

DNA fragments

[86]

Polymer

Drug

References

Bevacizumab

[57]

Serpin-derived peptide (SP6001)

[58]

Ranibizumab

[85]

Drug

References

Ranibizumab / Aflibercept

[60, 61]

(“hybrids”: microspheres containing nanoparticles) Poly(lactic-co-glycolic acid) (PLA nanoparticles entrapped into PLGA microspheres) Poly(lactic-co-glycolic acid) (Poly(beta-amino)ester, PBAE, nanoparticles entrapped into PLGA microspheres) Poly(lactic-co-glycolic acid) (Chitosan or chitosan-N-acetyl-L-cysteine based nanoparticles entrapped into PLGA microspheres) Polymer (“hybrids”: gel containing microspheres) Poly(lactic-co-glycolic acid) (Microspheres suspended into poly(N-isopropylacryl amide) gel

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In addition, microspheres can be used for the personalized medicine. In fact, the suitable drug dose needed by each patient can be obtained by simply adjusting the volume of the microspheres to be injected, according to the therapeutic window, the intravitreal drug pharmacokinetic, the drug payload in the microspheres and its release kinetics as well as patient needs [23]. 3.1. Poly(Lactic Acid) / Poly(Lactic-Co-Glycolic Acid) Poly(lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA) are biodegradable polymers/copolymers belonging to the group of polyesters. They contain hydrolysable bonds: the degradation by non-enzymatic hydrolysis of the ester backbone takes place in aqueous environments, such as the body fluids. They are therefore in vivo hydrolyzed into the safe monomers lactic and glycolic acids [24]. Physicochemical properties of PLA and PLGA vary depending on the molecular weight, resulting in solubility and viscosity changes. The process of polymer degradation depends on molecular weights and chemical compositions (relative ratios between the two monomers in the case of copolymers) [24]. They are commonly used for the preparation of microparticulate delivery systems using different technological methods: emulsification followed by solvent evaporation/solvent extraction and spray-drying are the most important processes used [25]. These polymers have been approved for clinical uses by U.S. Food and Drug Administration (FDA) and by European Medicines Agency (EMA). After their direct injection in the target, these systems release the loaded drugs over a long period of time (which depends on polymer characteristics) and then undergo degradation. The drug release can occur by diffusion through and/or erosion of the polymeric network. Drug diffusion can occur even before the degradation process of the polymeric network, while drug release through erosion occurs during the degradation process that determines the erosion of the polymer. The prevalence of diffusion with respect to erosion as drug release mechanisms mainly depends on Mw and solubility of the drug [24]. The first, isolated examples in literature appeared at the beginning of 90’s: PLGA and PLA microspheres containing fluorescein were injected intravitreally in rabbits [26], and biodegradable microspheres containing Adriamycin were proposed for the treatment of proliferative vitreoretinopathy [27]. At the end of 90’s, there is an increasing number of papers on the use of microspheres and these papers deal with a topic of interest: intravitreal administration of acyclovir (antiviral drug). Acyclovir is used for the treatment of acute retinal necrosis and herpes simplex virus retinitis, pathologies frequent in HIV positive immune competent patients. The systemic (intravenous) therapy is effective but it has deleterious side effects due to the high drug doses that must be done to overcome blood-ocular barrier. The intravitreal administration of PLA/PLGA microspheres is therefore proposed as an alternative to the systemic route of acyclovir. PLA and PLGA microspheres containing acycloguanosine (acyclovir) and designed for the intravitreal administration have been prepared using a spray-drying technique [28]. This technique is easy to use because it involves the preparation of a solution in which the drug and the polymer are dissolved

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and that is sprayed through the nozzle of a spray-dryer to achieve microspheres. The particle size of the spray-dried microspheres strongly depended on the Mw of the polymer used and it was in the range of 5-10 m for the lower Mw and around 20 m for the higher Mw used. In vivo evaluation was performed on albino rabbits: the drug concentration in the vitreous humour, due to drug released from microspheres, was initially high, higher than the concentration achieved by the intravitreal administration of an isotonic solution of the drug (as a comparison). The concentration of the drug was kept constant for fourteen days after administration of the suspension of microspheres. On the contrary, drug concentration decreased and three days after the administration of the drug solution it was no more detectable. Another paper reports acyclovir loaded PLGA microspheres which were obtained by emulsification/solvent evaporation method, using an oil-in-water (o/w) emulsion, with several additives [29]. Best results were obtained adding gelatin to the external phase of the emulsion. The authors described then a sterilization process of the intravitreal microspheres by -irradiation at a dose of 25 kGy [30]. The microspheres showed no change of their characteristics after -irradiation, mean diameters of non-sterilised (about 46 m) and sterilized (about 45 m) microspheres were not significantly different, demonstrating that the sterilization method, in the conditions applied, is suitable for the preparation of injectable microspheres. The same authors made a factorial design study [31], which resulted in a useful tool for the optimization of acyclovir PLGA microsphere preparation. An association of acyclovir and vitamin A palmitate loaded in PLGA microspheres is proposed by MartinezSancho et al. [32]. Vitamin A, both as free alcohol and esters, has shown to be effective by injection for the treatment of proliferative vitreoretinopathy, tractional retinal detachment and other vitreoretinal diseases [32, 33]. The best results were achieved with microspheres prepared with 40 mg of acyclovir, 80 mg of Vitamin A palmitate and 400 mg of PLGA. The microspheres were in vitro characterized: they showed a constant release of acyclovir and vitamin A for 49 days, while the injectability of a suspension of microspheres in isotonic saline solution resulted appropriate for its injection through a 27G needle. PLGA microspheres for intraocular administration of guanosine have been prepared by Chowdhury and Mitra [34]. Guanosine has been chosen as a model drug for its structural and solubility characteristics which are similar to those of acyclovir and ganciclovir, two important antiviral agents. The microspheres were obtained using a solventevaporation method, with oil-in-water (o/w) emulsions (particle size range of 70-160 m). The release of guanosine in phosphate buffer from PLGA microspheres occurs in a biphasic way. An initial burst effect (due to the drug absorbed onto the external surface of the microspheres) in the first 24 h, followed by a constant release that lasts until 100 h and more (which depends on the part of drug entrapped in the polymeric matrix structure of the microspheres). An in vitro/ex vivo experiment has been described to test the release behaviour of the microspheres. This test involves the use of the vitreous fluid of New Zealand albino rabbits. Vitreous fluid (1.5 ml) was removed from the eye globe of male


New Zealand albino rabbits using a tuberculin syringe; microspheres were dispersed in 1 ml of pH 7.4 isotonic phosphate buffer and vortexed for 1 min and one hundred microliters of microsphere suspension was then added to the Eppendorf tubes containing 1.5 ml vitreous fluid. Drug release in vitreous fluid (1.5 ml) was in the range of 6 to 10% in 10 h, depending on the polymeric composition of PLGA (Mw) used. As previously reported, Xu et al. [19] claimed that vitreous gel is structurally fragile and there is a liquefaction quickly after removal from the eye. In the light of these observations, we are wondering about the rationale of such in vitro/ex vivo tests. In another work, 5-fluorouracil (5-FU) loaded PLA/PLGA microspheres were prepared with a solvent/evaporation method [35]. The microspheres as a suspension were injected into the vitreous cavity of rabbits; it was reported that they were cleared from the vitreous cavity by 48 days. The histological study showed no abnormal findings after the injection. Intravitreal microspheres are also proposed to carry corticosteroids. Therapies with corticosteroids are frequently used in a broad spectrum of ocular inflammatory conditions [36]. Non-infectious uveitis is treated with corticosteroids and it is a good example of the problematics connected with the administration of a drug to the posterior segment of the eye. The systemic route represents often an initial treatment of choice of corticosteroids for non-infectious uveitis, but systemic adverse reactions can occur while topical delivery of steroids often fails to provide therapeutic levels into the vitreous cavity [37]. Intravitreal injections of steroids permit to obtain transitory therapeutic levels of the drug, without the side effects connected with systemic treatment. However, the clearance of corticosteroids from the posterior segment of the eye is fast, and repeated injections are needed to maintain the therapeutic levels of the drug [38]. Frequent intravitreal injections of steroids can cause vitreous hemorrhage, retinal detachment, or endophthalmitis and this risk increases with the number of injections [39]. Barcia et al. prepared PLGA microspheres loaded with dexamethasone [40], a glucocorticoid with antiinflammatory action, used as a treatment of choice in diseases that involve ocular inflammation [41]. The microspheres were prepared from an O/W emulsion by a solvent evaporation method and then sterilized by gamma irradiation (25 kGy), diameters were 20-53 m. Microspheres were injected into the vitreous of New Zealand rabbits (2.5-3.0 kg) affected by Panuveitis induced by intravitreal injection of Escherichia coli. The microspheres loaded with dexamethasone effectively reduced the short- and long-term intraocular inflammation in rabbit eyes. The authors put into evidence that such microsized drug delivery systems are characterized by an ability to treat not only the acute process of the pathology but also to diminish recidivism. Further studies involving intravitreal implants confirmed that the prolonged release of dexamethasone is the optimal choice for the treatment of chronic diseases affecting the posterior segment of the eye [42]. Villanueva et al. prepared biodegradable PLGA (50:50) microspheres loaded with dexamethasone and vitamin E [43]. Vitamin E is a natural antioxidant that can be used in


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the prevention and treatment of eye diseases [44]. The microspheres prepared (size around 20-40 m) showed the ability to control drug release for prolonged periods of time and were stable under standard refrigerated storage conditions. A preliminary injectability test carried out showed that microspheres were suitable for intravitreal injection with 30 G needles. Triamcinolone acetonide loaded PLGA microspheres were prepared by the solvent evaporation method and in vivo tested on a rabbit model of uveitis [45]. The microspheres containing triamcinolone acetonide (named RETAAC system) were intravitreally injected in human eyes for the treatment of macular edema and showed a good tolerance [46]. PLGA microspheres are prepared by Tanetsugu et al. for the controlled intravitreal release of ranibizumab [47]. This drug is a monoclonal antibody fragment against vascular endothelial growth factor (VEGF)-A; it is utilized for the therapy of age-related macular degeneration (AMD) caused by angiogenesis. It is characterized by a short half-life in the eye due to its low molecular weight and susceptibility to proteolysis. A ranibizumab biosimilar was incorporated into microspheres of PLGA to achieve both protection from enzymatic degradation and prolong drug release over several weeks. Therefore, PLGA microspheres can be proposed as a prototype platform for sustained drug delivery to the eye after IVT injection. Prolonged release of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) improves the survival and regeneration of neurons, but their systemic administration is characterized by possible side effects. For this reason, PLGA microspheres containing 4-(3-chloroanilino)6,7-dimethoxyquinazoline (EGFR TKI) were prepared and used in a rat optic nerve crush injury model [48, 49]. An oilin-water with co-solvent single emulsion solvent evaporation technique was used as preparation method of the microspheres [48]. Two weeks after intravitreal delivery microspheres promoted optic nerve regeneration [49]. The authors prepared also nanoparticles as a comparison and they found quite unexpected that after an initial burst effect the nanoparticles show slower release kinetics compared to the microspheres [49]. According to the authors, this behavior and in particular the release profiles longer-than-expected in the case of the nanospheres could be due to the fact that the polymeric network of the microspheres could have a less rigid structure allowing for greater access of water to the inner structure of the spheres and thus faster release, and vice versa for nanospheres. A severe disease of the posterior tract of the eye is glaucoma, which belongs to a group of neurodegenerative diseases that affects the optic nerve [50]. Neuronal damage can occur by different pathways, which lead to cell death with loss of retinal ganglion cells (RGCs) [51]. Checa-Casalengua et al. prepared PLGA microspheres containing the glial cell line-derived neurotrophic factor (GDNF) and vitamin E, for intraocular administration [52]. GDNF enhances the survival of neurons in neurodegenerative diseases; antioxidants such as vitamin E (-tocopherol) are also beneficial in neurodegenerative diseases. Furthermore, vitamin E has antiproliferative properties that might reduce some of the side effects typically linked to repeated intravitreal injections such as


proliferative vitreoretinopathy and retinal detachments. Starting from these considerations the authors prepared GDNF/vitamin E loaded PLGA microspheres able to give in vitro drug release of the bioactive for up to 19 weeks. Microspheres were prepared using an emulsification / solvent evaporation technique. This formulation was intravitreally injected into rats with elevated IOP (an animal model of glaucoma): it effectively protected the RGCs for at least 11 weeks. In a further work [53], the authors studied the GDNF levels after a single intravitreal injection of GDNF/vitamin E microspheres and pharmacokinetic studies were made in rabbits. The microspheres having particle size of about 19 m were suitable for injection as a suspension through 30-32 gauge needles. They found that a single injection of GDNF/vitamin E microspheres provided a sustained controlled release of the neurotrophic factor for a period of at least 6 months. The six-month follow-up after administration showed that the injections of GDNF/VitE loaded microspheres did not lead to any abnormality in the eyes treated. There were no reactions in correspondence of the anterior chamber in all animals. IOP levels remained within normal limits during the period studied (no case was over 14 mm Hg value). Also, Jiang et al. [54] showed that the IVT administration of PLGA microspheres containing GDNF significantly enhanced the survival of RGCs and their axons in a rat model of glaucoma, suggesting that GDNF delivered by PLGA microspheres can be an important neuroprotective tool for the treatment of glaucomatous optic neuropathies. Isiegas et al. prepared PLGA microspheres containing proinsulin and insulin which have shown their neuroprotective effects in several models of retinitis pigmentosa (RP) models [55]. They prepared the microspheres using a W1/O/W2 double emulsion followed by solvent evaporation. The microspheres were able to release the drug for several weeks in vitro. The in vivo results of this study showed that intravitreal administration of proinsulin loaded in PLGA microspheres determines neuroprotective effects in the rd10 mouse model used as the retinal degeneration in the rd10 mouse was slowed by a single IVT injection. Shelke et al. [56] studied the preparation of PLA microspheres for sustained IVT delivery of TG-0054, a watersoluble anti-angiogenic drug that can be used for the treatment of choroid neovascularization. The authors used PLA instead of the more hydrophilic PLGA because the drug is freely soluble in water and their goal was to prepare a formulation able to release the drug for several months. A waterin-oil-in-water emulsion/solvent-evaporation method was used. The in vivo intravitreal delivery of TG-0054 from microspheres was made in male New Zealand white rabbits. The microspheres showed good syringeability and they were administered through a small gauge needle. The microspheres provided an in vitro sustained drug release for 6 months and in vivo for about 3 months. Some researchers applied a combination of micro and nanotechnologies to prepare “hybrid systems” made of nanoparticles loaded into microspheres. For example, PLA nanoparticles entrapped in porous PLGA microspheres were prepared to obtain an intravitreal system able to release bevacizumab for a prolonged period of time. The release

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occurred for four months during the in vitro tests, while in vivo the drug was detectable until two months [57]. In an interesting paper by Shmueli et al. [58] nanoparticles containing a serpin-derived peptide (SP6001) and constituted by a poly(beta-amino ester) (PBAE) were loaded into PLGA microspheres. SP6001 belongs to a new class of peptides with antiangiogenic properties that can be used for the therapy of patients with neovascular age-related macular degeneration (NVAMD), a disease in which angiogenesis leads to the loss of central vision. SP6001 is a peptide with negatively charged glutamic acid residues and it interacts with a cationic polymer, such as PBAE, to achieve nanoparticles. A mouse model of choroidal neovascularization was used. The authors demonstrated that this polymeric delivery system can be potentially useful for the therapy of this macular degeneration. The strategy of complexing the peptide with PBAE to obtain nanoparticles and then encapsulating them into PLGA microspheres represented a successful strategy to obtain inhibition of angiogenesis for at least 14 weeks with a single intravitreal injection. This is a good example of a combination of technologies involving the preparation of nanoparticles and microspheres. Polyethylene-poly(lactic acid) (PEG-PLA) blockcopolymer (PEG(1000)-PLA(5000)) (the numbers in parentheses correspond to the molecular weights (MW) of each segment) was used by Rafat et al. [59] to prepare microspheres containing Tat-EGFP to be delivered to the retinal cells. Tat is an 11 amino acid domain of HIV-1 transactivator of transcription protein. It has been fused to EGFP to permit cellular internalization of the protein by micropinocytosis for the therapy of retinal diseases. A 22 factorial experimental design was used to prepare four PEG-PLA microparticulate formulations. The formulation with the best particle size and performances regarding protein release was then tested in culture using the 661 W cell line (retina-derived). This research showed that PEG-PLA microspheres are able to release Tat-EGFP without any cytotoxic effects to the retinal outer segment. The authors propose this system for subretinal injections but the results obtained in their study show that microspheres made of PEG-PLA block copolymers can be used for IVT route. Recently interesting studies have been published describing formulations constituted by drug-loaded PLGA microspheres suspended into an injectable hydrogel. This is to overcome the possible problem that after the intravitreal injection microspheres could move independently determining ocular complications. Osswald et al. prepared ranibizumab and aflibercept (anti-VEGFs) PLGA microspheres [60, 61]; hydrogels composed of poly(N-isopropylacrylamide) (PNIPAAm) were synthetized: they are hydrogels injectable at room temperature and able to transform into a viscoelastic structure at the physiological temperature. The hydrogel prepared can be injected using a 28G needle. In vitro release profiles were carried out in phosphate buffer: the microparticulate drug delivery system was able to release ranibizumab and aflibercept for 196 days with an initial burst in the first day. An animal (murine) model of choroidal neovascularization (CNV) has been made determining laser photocoagulation. Five to six lesions per eye were induced. The group treated with the anti-VEGFs loaded suspensions had


significantly smaller (60%) CNV lesion areas than the nontreated group. The research demonstrates that this kind of delivery system effectively decreases laser-induced CNV lesions using the murine model. 3.2. Poly--Hydroxybutyrate Many papers involve the use of PLGA and PLA, however also other polyesters can be used for the preparation of intraocular microspheres. Poly--hydroxybutyrate (PHB) microspheres were prepared by Hu et al. [62] loading doxorubicin, an anthracycline antibiotic which has been proposed for the IVT treatment of experimental proliferative vitreoretinopathy (PVR). PHB is a biodegradable polymer [63]. Doxorubicin-loaded microspheres were prepared by an emulsification/solvent evaporation method. A pharmacokinetics study was made using New Zealand rabbits. The results obtained showed that PHB microspheres are able to determine a more prolonged drug release than the IVT administration of a free drug solution. The study provided also evidence of the safety of doxorubicin-loaded PHB microspheres and demonstrated its utility for the IVT treatment of proliferative vitreoretinopathy. 3.3. Poly(Ester Amide) Poly(ester amides) (PEAs) are biodegradable polymers based on -amino acids, aliphatic dicarboxylic acids and aliphatic - diols, with ester and amide groups in the polymeric chain. They are block-copolymers with three types of building blocks randomly distributed along the polymer chain. The chemical structure of the polymeric backbone determines the biodegradability of the structure. These materials are characterized by good biocompatibility also in ophthalmic applications: PEA fibrils were prepared by extrusion and have been used in an animal (rabbit) experimental model for subconjunctival and IVT routes [64]. Andrés-Guerrero et al. prepared PEA microspheres loaded with dexamethasone (chosen as a lipophilic model drug) using an emulsion solvent-evaporation technique [65]. The microspheres can be readily sterilized by gamma irradiation (a dose of 25 kGy was applied) and administered as a suspension using 27-32G needles. The microspheres had a mean particle size ranging from 10 to 20 m, suitable to be injected as suspensions through standard needles (27-34G). No differences in morphology, drug content and drug release behaviour were found before and after the sterilization process of the microspheres. In vivo studies showed that the microspheres release dexamethasone in rabbit eyes for up to 3 months. 3.4. Porous Silicon Porous silicon is a biodegradable material characterized by porosity and consequently remarkable surface area which make it suitable for the preparation of drug delivery systems (400-800 m2/g) [66]. Porous silicon is also characterized by a good ocular biocompatibility [67]. Furthermore, the surface of this material can be functionalized by hydrosilylation, oxidation and silanization to permit the loading of drugs by chemical bonds and to increase its vitreous residence time [67, 68]. Oxidized porous silicon particles covalently grafted with daunorubicin have been prepared by Chhablani et al. as a sustained intraocular drug delivery system obtaining a pro-


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longed IVT drug release without any ocular toxicity [68]: daunorubicin is a potent cell growth that can be used to treat proliferative vitreoretinopathy (PVR). Intravitreal controlled release of dexamethasone has been achieved from engineered microspheres made of porous silicon dioxide by Wang et al. [69]. In vitro drug release studies showed that dexamethasone was released from silicon particles for a long period of time, over 90 days. In vivo studies showed that no intraocular adverse reactions occur in rabbit eyes after a single 3 mg IVT injection and that free drug level at 2 weeks after IVT administration was well above the therapeutic level. 3.5. Chitosan Chitosan is an amino polysaccharide (poly-1,4-Dglucoamine) obtained from chitin by deacetylation [70, 71]. Chitin is a component of the exoskeleton of insects and shellfish and it is one of the most abundant polysaccharides present in nature. Chitosan is a biodegradable, biocompatible polymer widely used in many biomedical applications [7274]. This polymer is a good drug carrier to deliver drugs through different routes including the ocular administrations [75-77]. It is also reported that it can inhibit the proliferation of fibroblasts [78] and that in glaucoma filtration surgery chitosan reduces postoperative scar formation and improves surgical effectiveness [79, 80]. Yang et al. proposed chitosan as a filling material of vitreous body to inhibit proliferative vitreous retinopathy [81]: the authors demonstrated that chitosan used in intravitreous buffer showed no fluctuation of intraocular pressure and no severe inflammatory responses in intraocular tissues were found, showing it to be a promising material for medical applications in the vitreous cavity. Chitosan, moreover, is known to determine electrostatic interactions with negatively charged surfaces of epithelial cells [82]. Chitosan has gained attention as possible gene carriers, also in the ocular field [83]. The same group that prepared PEG-PLA microspheres for the release of Tat-EGFP, previously cited [59], developed chitosan microspheres for the delivery Tat-EGFP to the retina [84], to improve the amount of the protein released from the microparticles. Tat-EGFP loaded microspheres were tested for cellular toxicity in photoreceptor-derived 661W cells. They presented no in vitro cellular toxicity at low concentrations (up to 1 mg ml-1), but at a higher concentration of 10 mg ml-1 they showed cytotoxic effects. This research showed that chitosan microspheres are effective long-term protein for drug delivery carriers that can be effectively used for the treatment of the posterior segment of the eye. The polymer (chitosan) concentration can determine cytotoxicity. In fact, the authors report that chitosan microspheres at high concentrations could be toxic to retinal cells. However, as it was difficult to determine the exact concentration of chitosan exposed to individual cells, it was difficult to make definitive conclusions. Some “hybrids” were prepared by Elasiad et al. in which chitosan-based nanoparticles were entrapped into PLGA microspheres preparing “system-within-system” that can be proposed for the IVT delivery of ranibizumab [85]. Chitosan-based nanoparticles made of chitosan or chitosan-Nacetyl-L-cysteine (size 17-350 nm) were incorporated into


PLGA microspheres with a w/o/w (double) emulsion technique. Ultrapure oligochitosans have been studied and proposed as DNA carriers in ocular tissues such as the retina by Puras et al. [86]. Oligochitosan based polyplexes were prepared and characterized to evaluate the transfection efficiency in rat retinas after subretinal and IVT administrations. IVT injections transfected cells in the inner nuclear and plexiform layers of the retina and mainly in the ganglion cell layer. CONCLUSION Intravitreal administration of drugs is an invasive technique that is however indispensable for the treatment of many pathologies of the posterior segment of the eye. Owing to the rapid clearance of therapeutics from the vitreous body there is the need of formulations able to release the drug for a prolonged period of time. Different kind of formulations can be proposed for intravitreal use such as biodegradable micro and nanoparticles. Nowadays the use of nanomedicine is a field with increasing importance, however in the case of intravitreal administrations microspheres are still important. Microspheres are easy to prepare and manage, and good results in terms of prolonged drug release and improvement of drug stability can be obtained. It has been reported that in some cases larger particles can remain in the vitreous body for longer periods of time owing to their lower mobility. Some discordant results are present in the literature regarding a correlation between particle size and time of residence into vitreous but this can be due to the fact that in the case of nanoparticles besides the size, the surface characteristics of the particles play an important role, while in the case of microparticles steric considerations can be prevalent. Despite “nanoparticle era” microspheres are still important dosage forms in the field of intravitreal systems. They do not have to be considered in competition with nanomedicine but as an alternative or in some cases (“hybrid systems”) a way by which nanoparticles can even improve their properties. CONSENT FOR PUBLICATION

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The authors declare no conflict of interest, financial or otherwise. ACKNOWLEDGEMENTS

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