Chitosan-based nanostructures: A delivery platform for ocular therapeutics

Advanced Drug Delivery Reviews 62 (2010) 100–117 Contents lists available at ScienceDirect Advanced Drug Delivery Reviews j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / a d d r Chitosan-based nanostructures: A delivery platform for ocular therapeutics☆ Maria de la Fuente b, Manuela Raviña a, Patrizia Paolicelli a, Alejandro Sanchez a, Begoña Seijo a, Maria Jose Alonso a,⁎ a b Department of Pharmacy and Pharmaceutical Technology, University of Santiago de Compostela, Faculty of Pharmacy, Campus Sur, 15782 Santiago de Compostela, Spain Department of Pharmaceutical and Biological Chemistry, University of London, The School of Pharmacy, WC1 1AX London, UK a r t i c l e i n f o Article history: Received 1 July 2009 Accepted 10 November 2009 Available online 1 December 2009 Keywords: Chitosan Ocular Ophthalmics Drug delivery Gene delivery Hyaluronan Nanocapsules Nanoparticles Nanostructures Emulsions a b s t r a c t Nanoscience and nanotechnology has caused important breakthroughs in different therapeutic areas. In particular, the application of nanotechnology in ophthalmology has led to the development of novel strategies for the treatment of ocular disorders. Indeed, the association of an active molecule to a nanocarrier allows the molecule to intimately interact with specific ocular structures, to overcome ocular barriers and to prolong its residence in the target tissue. Over the last decade, our group has designed and developed a delivery platform based on the polysaccharide chitosan, which suits the requirements of the topical ocular route. These nanosystems have been specifically adapted for the delivery of hydrophilic and lipophilic drugs and also polynucleotides onto the eye surface. The results collected up until now suggest the potential of this delivery platform and the subsequent need of a full preclinical evaluation in order to satisfy the specific regulatory demands of this mode of administration. © 2009 Elsevier B.V. All rights reserved. Contents 1. 2. 3. 4. 5. 6. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Critical barriers in ocular therapeutics . . . . . . . . . . . . . . . . . . 2.1. Critical barriers for classical low molecular weight compounds . . . 2.1.1. Lipophilic drugs . . . . . . . . . . . . . . . . . . . . 2.1.2. Hydrophilic drugs . . . . . . . . . . . . . . . . . . . 2.2. Critical barriers for gene-based therapies . . . . . . . . . . . . . Chitosan solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . Chitosan-based delivery vehicles for small molecules . . . . . . . . . . 4.1. Chitosan-based nanoemulsions and nanocapsules . . . . . . . . . 4.2. Chitosan coated nanocapsules and nanoparticles . . . . . . . . . 4.3. Chitosan nanoparticles . . . . . . . . . . . . . . . . . . . . . 4.4. Chitosan/cyclodextrin nanoparticles . . . . . . . . . . . . . . . Chitosan-based delivery vehicles for gene therapies . . . . . . . . . . . 5.1. Chitosan–DNA complexes . . . . . . . . . . . . . . . . . . . . 5.2. Chitosan nanoparticles . . . . . . . . . . . . . . . . . . . . . 5.3. Chitosan/hyaluronic acid nanoparticles . . . . . . . . . . . . . . 5.4. Chitosan/alginate nanoparticles . . . . . . . . . . . . . . . . . 5.5. Chitosan–lipid complexes . . . . . . . . . . . . . . . . . . . . Biodistribution, toxicity and biocompatibility . . . . . . . . . . . . . . 6.1. Biodistribution: interaction of Chitosan-based nanoparticles with the . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ocular epithelia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Chitosan-Based Formulations of Drugs, Imaging Agents and Biotherapeutics”. ⁎ Corresponding author. Tel.: +34 981 563100(14885); fax: +34 981 547148. E-mail address: mariaj.alonso@usc.es (M.J. Alonso). 0169-409X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2009.11.026 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 101 103 103 104 104 104 106 107 107 108 108 109 109 109 109 110 110 111 111 101 M. de la Fuente et al. / Advanced Drug Delivery Reviews 62 (2010) 100–117 6.2. Toxicity and biocompatibility 6.2.1. In vitro studies . . . 6.2.2. In vivo studies . . . 7. Conclusions . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction There are a number of challenges associated to the treatment of ocular diseases. In general, the major problem in ocular therapeutics is to maintain an effective drug concentration at the site of action for an appropriate period of time, in order to achieve the expected pharmacological response [1]. Ophthalmic drug delivery, probably more than any other route of administration, may benefit from the characteristics of nanotechnology-based drug delivery systems [2]. The use of nanocarriers provides interesting opportunities for topical ocular drug delivery, mainly because of their capacity to protect the encapsulated molecule while facilitating its transport to the different compartments of the eye [3–9]. Additionally, nanostructures may offer the possibility of controlling drug delivery, thus being attractive vehicles for the treatment of some chronic ocular diseases [7,10,11]. Furthermore, nanocarriers are critical in order to exploit the emerging field of new gene therapies for the treatment of ocular disorders [12,13]. Other alternatives for topical drug delivery involve the use of liposomes, microemulsions and microparticles, among others [10,14–21]. Over the last two decades we have dedicated significant efforts to the rational development of ocular drug delivery nanostructures. More precisely, our activity in the ocular drug delivery field started with the development of hydrophobic nanocarriers consisting of polyalkylcyanoacrylate [22–24] and polyesters, particularly poly-ε caprolactone [25–27]. This initial work led us to the conclusion that these nanocarriers have an affinity towards the corneal epithelium and also suffered a certain aggregation upon contact with the mucosal surface. In a second stage, our goal was to provide the nanocarriers with a hydrophilic coating intended to improve their stability and their interaction with the mucosa. For this purpose, we selected hydrophilic materials i.e. polyethyleneglycol (PEG) and chitosan (CS) [28,29]. PEG was selected because of its well-known protein rejecting properties (shielding effect), whereas the choice of CS, a cationic polysaccharide, was mainly justified by its mucoadhesive and penetration- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 113 113 114 114 enhancing properties, as well as by its good biocompatibility with the ocular structures [30–33]. The very promising results obtained with CS-coated systems led us to the further development of nanostructures made of solely hydrophilic polysaccharides such as CS alone or in combination with cyclodextrins and hyaluronic acid [34–36]. The evolution in the design of these nanostructures is highlighted in Fig. 1. As will be later described, the versatility of the designed nanostructures has allowed us to properly formulate hydrophilic and lipophilic drugs [6,34] as well as genetic material [37,38]. In this review, we summarize the current advances made on CS nanostructures specifically designed by our group and others for the topical delivery of a variety of therapeutic drugs to the eye. In order to provide the reader with a clear overview of the potential of these vehicles we have classified them in different sections depending on their utility for the delivery of either lipophilic drugs or gene molecules. Additionally, we present critical data with regard to their action mechanism and to toxicological issues. 2. Critical barriers in ocular therapeutics Topical instillation of an active compound is the first method choice of delivery in ocular therapy. However, due to the innate protective characteristics of the eye against the entry of foreign compounds, the bioavailability of an instilled compound is generally low. The eyeball consists of two anatomical regions: the anterior segment, in which the cornea and conjunctiva are the main prominent structures, and the posterior segment, in which the retina plays the most important function (i.e. transduction and adaptation to different levels of light) [17]. The cornea is a non-vascularized barrier consisting of five to seven layers, which exhibits high resistance to passive diffusion of ions and molecules and withstands the intraocular pressure [39]. This tissue has a smaller surface area compared to the conjunctiva which, moreover, is a leakier epithelium than the cornea [40]. Traditionally the role of the conjunctiva has been considered to be mainly protective and functioning as a passive physical barrier. Fig. 1. Relevant findings, in a time line course, regarding the application of CS nanostructures in the management of ocular diseases. 102 M. de la Fuente et al. / Advanced Drug Delivery Reviews 62 (2010) 100–117 However, nowadays it is known that there are several transporters (e.g. P-glycoprotein, amino acid, etc) which play a critical role in achieving influx and efflux transport of drugs in the conjunctiva. As a consequence, the feasibility for intraocular drug delivery via the conjunctival route is now well documented [17,40]. Nevertheless, it should also be taken into account that the presence of lymphatic and blood vessels can lead to significative systemic absorption [41]. Covering both corneal and conjunctival surfaces and forming part of the tear film, is a mucus layer, which is secreted by the goblet cells of the conjunctiva. The lachrymal film plays a multifunctional role, since it hydrates, cleanses, lubricates and serves as a defense against the pathogens; but also, it involves an additional obstacle to any drug penetration [42]. Moreover, the lachrymal film is a dynamic fluid that undergoes a constant renewal and therefore limits the time of residence of the drugs on the surface of the eye. In addition to the physical barriers, ocular tissues contain metabolic enzymes, such as esterases, aldehyde and keton reductases [43], which may degrade and reduce the efficacy of the drugs. As a result of these anatomical and physiological constraints [41,44] after topical application, a major fraction of the administered drug is lost by different mechanisms, resulting in very low ocular bioavailability (a schematic illustration can be seen in Fig. 2). Altogether, for the successful treatment of pathologies that affect the posterior segment of the eye, improving the corneal and/or conjunctival permeability becomes one of the main challenges in ocular drug delivery. Alternative routes other than topical, i.e. the systemic route or intrachameral injections, have also been considered for increasing the bioavailability of drugs in the internal structures of the eye. In the case of systemic administration, a major drawback is that only 1–2% of the administered drug reaches the vitreous cavity, Fig. 2. Schematic illustration of the critical biological barriers that drugs need to overcome after topical administration onto the eye surface (A), and specific intracellular barriers for effective nucleic acid delivery (B). 103 M. de la Fuente et al. / Advanced Drug Delivery Reviews 62 (2010) 100–117 especially impeded by the blood–retinal barrier which is selectively permeable to more lipophilic molecules and mainly governs the entry of drug molecules into the posterior segment of the eye [45]. This results in frequent administration of high amounts of drug leading to important systemic side effects. Other routes of administration are currently being used for the delivery of drugs to the posterior segment and particularly to the retina [18]. Some alternatives involve either injections of a drug, a drug delivery carrier or a drug delivery device into the vitreal cavity of the eye, or a periocular delivery following a trans-scleral route to the back of the eye that allows it to penetrate the retinal pigment epithelium [14]. Despite all the above described inconveniences of topical administration (Fig. 2A), non-invasive routes continue to be favored in the clinical practice. Thus, the development of topical ocular drug delivery systems with a potential of overcoming these constraints currently represents a promising approach for the treatment of ocular diseases. An evidence of the potential of nanostructured and other drug delivery formulations is illustrated in Table 1. Indeed, in 2002 the FDA approved the clinical use of an anionic emulsion containing cyclosporine A 0.05% (Restasis®, Allergan) for the treatment of chronic dry eye. A similar formulation (anionic emulsion containing difluprednate 0.05%, Durezol™, Sirion Terapeutics) has recently been approved for the treatment of ocular inflammation. In the same field, a non-medicated anionic emulsion for eye lubricating purposes, in patients suffering from moderate to severe dry eye syndrome (Refresh Dry Eye Therapy®, Allergan), and two lipidic emulsions, indicated for the restoration of the lipid layer of the lacrimal fluid (Lipimix™, Tubilux Pharma, and Soothe XP® Emollient, Bausch and Lomb), have been launched in the US and European markets. On the other hand, cationic nanoemulsions have also made their way onto the market. Namely, the product Cationorm® (Novagali Pharma, France) was launched in the European market for the treatment of dry eye symptoms and two more products, based upon the same technology and intended to deliver cyclosporine A, are currently under registration or under clinical evaluation (Phase III). A number of review articles have already described the ocular barriers and the main limitations for several drugs to reach their targets at the different ocular sites [9,14,41,46,47]. Therefore, in this review our interest is to briefly summarize these barriers in the case of classical low molecular weight drugs and to emphasize those which are relevant in ocular gene therapy. 2.1. Critical barriers for classical low molecular weight compounds 2.1.1. Lipophilic drugs Although conventional eye drops cannot be considered optimal in the treatment of ocular diseases, more than 90% of the marketed ophthalmic formulations are developed in this form. This type of formulation would represent a challenge in the case of lipophilic drugs. In fact, highly lipophilic drugs cannot be dissolved in an aqueous medium and need to be presented in the form of suspensions or emulsions. These formulations often suffer of stability problems and are not easily accepted by the patients. Moreover, the discomfort they cause in the patients may lead to blinking and, thus, to the loss of a significant amount of drug. Apart from this, the drug that remains on the ocular surface will have to partition or dissolve into the lacrimal fluid before it can diffuse through the corneal and epithelial barriers [48]. As indicated before, when the target site is located in the inner eye, the drug can follow two different pathways: the cornea and the conjunctiva. The cornea is a heterogeneous barrier composed of the corneal epithelium (a major hydrophilic barrier), the stroma (a major lipophilic barrier) and the endothelium (a minor lipophilic barrier). Because of this nature, lipophilic drugs can easily cross the corneal epithelium by a transcellular pathway (i.e. through the cells) either by facilitated transport or by diffusion through the lipid bilayer [49]. The former requires particular chemical interactions with transporters native to the cells, while the latter requires lipophilicity, which has been classically improved through the use of prodrugs or analogs [14]. After, the transport of the drug from the epithelium to the stroma may be hampered by the hydrophilic nature of the barrier, being this the main limiting step for the drug to access the inner eye. In fact, the stroma may act as a reservoir from which the drug will be slowly delivered to the aqueous humour. Finally, the endothelium is a very Table 1 Some current formulations approved or in advanced phase in clinical trials for ocular topical administration. Product Company Cationorm® Novagali Vekacia® Cyclokat® Lipimix® SootheXP® Emollient Durezol™ Restasis® Drug–treatment Drug free Mild dry eye syndrome Novagali Cyclosporine A Vernal keratoconjunctivitis in pediatrics Novagali Cyclosporine A Moderate to severe dry eye Tubilux Drug free Restores lipid layer of the lacrimal field following refractive or other ocular surgery Bausch & Lomb Drug free Restores lipid layer of the lacrimal field following refractive or other ocular surgery Sirion therapeutics Difluprednate Inflammation and pain associated with ocular surgery Allergan Refresh Dry Eye Therapy® Allergan Formulation Status Strength/recommended application Cationic emulsion Commercialized in France 1–4 times/day Cationic emulsion Under registration process – Cationic emulsion Phase III clinical trials – Phospholipid emulsion Comercialized in Italy, Germany and France 2–3 times/day Oil-based emulsion Commercialized in USA 1 to 2 drops as needed Anionic emulsion Commercialized in USA Cyclosporine A Anionic emulsion Increase tear production in patients whose tear production is presumed to be suppressed due to ocular inflammation associated with keratoconjunctivitis sicca Drug free Anionic emulsion Eye lubrication for moderate to severe dry eye syndrome Commercialized in USA Difluprednate 0.05% 4 times/day for two weeks after surgery followed by 2 times/day for a week and then a taper based on the response. Cyclosporine A 0.05% 2 times/day Commercialized in USA May be used as often as needed 104 M. de la Fuente et al. / Advanced Drug Delivery Reviews 62 (2010) 100–117 thin layer of cells which does not offer a significant resistance to drug transport [50,51]. With respect to the conjunctiva, this has gained increasing interest with regard to its implication in the access of drugs to the inner eye. The conjunctiva is a leakier epithelium than the cornea, therefore expected to have a higher permeability for large hydrophilic compounds that are transported via paracellular pathways. However, since lipophilic drugs mainly use a transcellular transport, the corneal route currently dominates [39,40,50]. Taking all these limitations and barriers as a whole, it could be concluded that, in order to enable a significant transport of the lipophilic drug to the inner eye, it is of primary importance to achieve an important accumulation of the drug in the corneal epithelium. This deposit of drug may represent the driving force for the significant and prolonged delivery of the drug to the inner ocular tissues. As will be later discussed, the use of nanovehicles may significantly help improving the tolerability of the formulation and also facilitate the accumulation of the drug in the protective epithelia. 2.1.2. Hydrophilic drugs Contrary to lipophilic drugs, hydrophilic drugs can be easily formulated as aqueous eye-drop solutions; however, they also suffer from a low residence time over the ocular surface. Moreover, hydrophilic drugs have great difficulties in passing from the tear film to the corneal/conjunctival epithelia. The paracellular entry through both corneal and non-corneal epithelia becomes the most important penetration route for hydrophilic drugs [52]. Nevertheless, the intercellular spaces in the cornea have tight junctions that serve as a selective barrier and, thus, this paracellular route is limited to very low molecular weight compounds [14,53–55]. As regards to the absorption by the non-corneal route, studies have conclusively proved that the conjunctiva is a leakier epithelium that plays an important role in the absorption of large hydrophilic molecules [39,40,50,56]. Overall, these very important barriers greatly limit the utility of hydrophilic drugs in ocular therapies. The classical attempts for improving the bioavailability of these drugs include the use of viscosity enhancers (e.g. cellulose derivatives) mucoadhesive polymers (e.g. polysaccharides), and in situ gel-forming systems [14]. However, the improvements achieved with these formulations have been certainly limited. Therefore, the use of nanocarriers able to entrap and deliver hydrophilic macromolecules into the ocular epithelia, also emerge as a promising strategy that will facilitate their use in the clinical practice [1,9]. 2.2. Critical barriers for gene-based therapies Polynucleotides are hydrophilic molecules and, therefore, the barriers described in the previous section also apply. However, their specific nature and target site makes their effective delivery a challenging task for researchers in the ocular drug delivery field. Indeed, because of their high molecular weight and negative charge, and also due to the fact that they have an intracellular/intranucleous target site, polynucleotides need to sort specific critical barriers. In the following paragraphs, we will provide information concerning these specific critical barriers. As mentioned before, topical delivery to the eye would be the most convenient way of ocular gene delivery. However the tear film is the first drawback for the low nucleic acid bioavailability, not only due to its high renewal rate, but also due to the presence of extracellular endonucleases that specifically degrade nucleic acids [57]. Moreover, as a direct consequence of their high molecular weight and negative charge, polynucleic acids are unable to pass across the corneal barrier and remain confined to the superficial epithelial layer [58]. In relation to the conjunctiva, some authors have suggested it as a more viable route for the ocular delivery of genes because of its higher permeability compared with the cornea [53,55]. However, the tight junctions that join epithelial conjunctival cells still limit the paracellular transport of nucleic acids. Taking into account this low nucleic acid permeability, several authors have explored alternative routes for overcoming these epithelial barriers, such as intracorneal or intraconjunctival injection. Nevertheless, the results have shown that nucleic acids remain confined to the area of the injection and transfect only the periocular tissues at the injection site [59]. In some cases the target site of nucleic acids may be located in the posterior segment of the eye (retina, vitreous, choroid) [13,60]. Two modalities of administration, namely subretinal and intravitreal injection have been explored for reaching these target tissues. Intravitreal injection, which is less invasive than subretinal injection, is normally preferred. The main limitation of the intravitreal route is the very low intravitreal half-lives of nucleic acids, thus requiring repeated intraocular administrations, which might lead to lens damage and retinal detachment. For that reason, the use of sustained delivery systems has been regarded as a promising strategy of prolonging the nucleic acids stability in the vitreous. A specific requirement for these delivery systems is their adequate diffusion through the vitreous humour [61–63]. Vitreous is a greatly hydrated (98% water) gel-like material consisting mainly of collagen, hyaluronan, and proteoglycans containing chondroitin sulfate and heparan sulfate. Consequently, this specific environment itself represents an important challenge in the design of intravitreal controlled delivery carriers. Although overcoming the above mentioned extracellular barriers and reaching the target cells is a challenge itself for the ocular delivery of genes, intracellular barriers strongly hamper nucleic acids towards the target compartment/molecule. These barriers include traversing the cellular membrane, diffusing through the cytosol while escaping lysosomal degradation and, in some gene therapy applications, overcoming the nuclear envelope (Fig. 2B). With regards to the first barrier, the cellular membrane, it is a dynamic structure of lipophilic nature, which restricts the entry of large, hydrophilic, or charged molecules [64]. The negatively charged phosphate backbone of the nucleic acid molecule is the primary cause of its inadequate and inefficient cellular association, owing to electrostatic repulsion from the negatively charged cell surface. Consequently, only through the association to an appropriate delivery carrier, the genetic material will have a chance of effective cellular internalization. Upon endocytic internalization, nucleic acids along with their delivery vectors are compartmentalized into endosomal vesicles, where the genetic material can be inactivated or degraded. The endosome undergoes acidification to a pH of 5 to 6, which, in addition to promoting acidic hydrolysis, activates lysosomal enzymes that can rapidly degrade oligonucleotides or plasmids [65,66]. Escape from the endosome may leave the delivery system placed at some distance from where its action takes place. Transport of nanostructured matter through the cytoplasm to their target site is a vital but not trivial process [67]. Finally, when the target site is not located in the cytoplasm and requires nuclear entry, as happens in the case of plasmid DNA, an additional challenge is the nucleic acids transport through the nuclear pores [65,68]. Consequently, the design of improved drug delivery systems should help to overcome these anatomical and physiological barriers that act as rate-limiting steps in achieving an effective drug bioavailability. However, drug delivery systems should be designed to meet the specific requirements of their hydrophilic, lipophilic or nucleic acid nature. In the following sections we will describe the potential of CS for ocular drug delivery and how formulations have been adapted to meet their specific requirements. 3. Chitosan solutions A major challenge in ocular therapeutics is the improvement of ocular drug bioavailability. As indicated before, conventional aqueous M. de la Fuente et al. / Advanced Drug Delivery Reviews 62 (2010) 100–117 solutions topically applied to the eye have the inherent disadvantage that most of the instilled drug is lost within the first 15–30 s after instillation, due to reflex tearing and drainage via the nasolacrimal duct. Hence, many of the efforts at improving ocular drug delivery have been focused on increasing the duration of the drug contact time. The first step in this direction has been to enhance the precorneal retention of ophthalmic solutions by the incorporation of viscositybuilding agents such as polyvinyl alcohol and methyl cellulose [69]. However, viscosity has been found to have a minor consequence in prolonging the ocular residence time of drugs. Thus, over the last years, the use of mucoadhesive polymers has attracted significant attention for the achievement of this objective. The capacity of some polymers to adhere to the mucin coat covering the conjunctiva and the corneal surfaces of the eye by non-covalent bonds forms the basis of ocular mucoadhesion. Mucoadhesive polymers increase the drug residence time because the turnover of the mucus layer is very slow (approximately 15 to 20 h). Moreover, mucoadhesive polymers provide an intimate contact between the drug and the absorbing tissue, which normally results in a high drug concentration in the local area [70,71]. Chitosan (CS) is a cationic polysaccharide that has widely being used in ophthalmic preparations [30]. The specific biadhesiveness of CS to the ocular surface was first observed in an ex-vivo study, in which the activity of radiolabelled CS was measured by scintillation counting after addition to a freshly excised cornea and exhaustive rinsing [72]. Electrostatic attraction appears to be the major driving force for mucoadhesion [32]. However, both hydrogen bonding and hydrophobic interactions are also supposed to have a role in this process [73,74]. Even more so, the pH of the CS solution as well as the co-existence of other chemicals are known to affect the relative contribution of each physical interaction [74]. Apart from its mucoadhesive character, there are other favorable biological properties which render CS a very attractive polymer for ocular drug delivery. First at all, CS has penetration-enhancing properties and has attracted a lot of attention as a potential absorption enhancer across the mucosal epithelia. This fact has been mainly attributed to the opening of the tight junctions located between epithelial cells, resulting in an enhancement of the absorption via the paracellular route [33,75,76]. In addition, some authors have also reported the possibility of additional intracellular pathways which may contribute to the enhancement of the cellular permeability attributed to CS [77]. On the other hand, CS solutions show pseudoplastic and viscoelastic properties [78,79], desirable for ocular drug administration. Indeed, the pre-corneal tear film has a pseudoplastic character that should not be disturbed by the application of liquid formulations: blinking involves high rates of shear within the tear film, requiring a low tear viscosity to avoid damage to epithelial surfaces; conversely, in the open eye, a higher viscosity is desirable to resist drainage and film break-up [80]. As a consequence, pseudoplasticity is particularly important in ophthalmic formulations. It also facilitates the retention while it permits the easy spreading of the formulation due to the blinking of the eyelids [81]. A decade ago, Felt et al. reported that CS has an excellent ocular tolerance [31]. This affirmation was based on the results observed in a rabbit model following topical instillation of CS solutions and using confocal laser scanning ophthalmoscopy combined with corneal fluorescein staining [31]. Additional evidence of the low toxicity of CS solutions has been reported by Di Colo et al. [82]. In this study, the authors described the absence of apparent irritation signs, such as conjunctival/corneal edema and/or hyperemia following topical instillation of CS solution to rabbits. Besides its acceptable biocompatibility, a major advantage of CS relies on its biodegradability. Indeed, CS can be degraded by lysozyme [83,84], an enzyme that is highly concentrated in the lacrimal fluid (450 to 1230 mg/L) [85]. Moreover, in a very recent study, Gorzelanny et al. [86] have identified the role of other common 105 enzymes considered as part of the innate immune response, such as the human chitotriosidase, involved in CS degradation. In addition, the antibacterial activity of CS is an advantage for ocular drug administration. Although the precise mechanisms of antimicrobial action of CS are yet to be elucidated [87], there is a consensus about the interaction of CS with the negatively charged cell membranes, thus leading to the leakage of proteinaceous and other intracellular constituents. The antimicrobial activity of CS in the ocular surface was specifically corroborated by Felt et al., who proposed the use of a CS solution as an artificial tear formulation for the treatment of dry eye [88]. The rationale for choosing CS was based on its excellent tolerance after topical application and its ability to spread over the entire cornea. Moreover, the antibacterial properties of CS may represent an additional advantage towards this specific application, because of the frequency of secondary infections concurrent with dry eye pathologies. The simplest presentation of CS in a liquid formulation consists of a CS solution. Felt et al. [31] presented for the first time the ability of CS to increase pre-corneal drug residence time, using gamma scintigraphy. The presence of CS in an ophthalmic solution resulted in a significant increase of the precorneal residence time of tobramicyn and ofloxacin when compared with commercial drug solutions [31,89]. No influence of the type of the CS molecular weight or of its concentration in the formulation was observed. This led to the suggestion that the improvement in retention time of the drugs, when using CS, might not only be induced by an increase in the viscosity of the solutions, but also to a saturable bioadhesive mechanism [31]. Similarly, in our group we have observed a prolonged retention of a solution of fluoresceinlabelled CS, up to 24 h, following its topical administration to rabbits [5]. More recent studies performed by Di Colo et al. have also shown the ability of CS hydrochloride to facilitate the transcorneal penetration of ofloxacin into the aqueous humour of rabbit eyes [82]. This positive effect was attributed to an enhancement of the corneal permeability presumably linked to the polycationic nature of CS hydrochloride. In fact, in the same work, a derivative of CS that behaves as a polyanion at the physiological pH of the tear fluid, N-carboxymethylchitosan, failed to significantly enhance the intraocular drug penetration. Other strategies recently explored, in order to improve ocular drug bioavailability, are based on the derivatization or combination of CS with thermoresponsive polymers. For example, the chitosan–poly (N-isopropylacrylamide) derivative was found to exhibit in situ gelforming properties [90]. This thermosensitive gel-forming system led to a significant improvement of the ocular availability of timolol maleate, the area under concentration–time curve (AUC) being twofold greater than that obtained with the conventional drop solution. The thermosensitive system forms a gel after dropping onto the ocular surface, promoting a sustained release and prolonged pre-corneal retention of timolol maleate. In addition, the inherent capacity of CS as a penetration enhancer and its bioadhesive characteristics may play a role in promoting the corneal penetration of timolol. In a similar work, it was observed that by mixing the thermosensitive polymer Pluronic F-127 with CS, it was possible to induce a controlled gelation which led to a significant increase of the transport of timolol maleate across the corneal membrane [91]. Besides the increase in the bioavailability observed for small hydrophilic molecules when co-formulated with CS, attributed to the ability of CS to interact with the ocular mucosa and favor the transport of drugs through the corneal epithelia, the development of more sophisticated delivery systems, intended to the improved entrapment of lipophilic compounds and highly labile macromolecules such as nucleic acids, have gained an increasing interest over the last years [92]. Apart from the development of more conventional forms, such as implants or contact lenses, several nanoparticulate formulations have been investigated during the last few years and have exhibited a certain degree of success as ocular carriers for a variety of drugs, including therapeutic genes [93,94]. As disclosed in the following 106 M. de la Fuente et al. / Advanced Drug Delivery Reviews 62 (2010) 100–117 protect them from the harmful biological environment [9,11,94]. Depending of their composition and structure, these nanovehicles may offer the additional possibility of controlling the release rate of the associated drug [7]. Within the frame of the ophthalmic application, the emphasis has been made on the design of nanocarriers for improving the ocular bioavailability of poorly watersoluble molecules. This is important because a wide range of drugs currently intended for the treatment of different extra- and intraocular diseases such as many antibiotics, anti-inflammatory and antiglaucoma drugs, have a more or less important hydrophobic character. Moreover, a high percentage of newly discovered drugs have limited solubility in an aqueous media and many will never reach the clinical use due to formulation problems [97]. Within this particular context, nanovehicles represent an interesting alternative to oily conventional formulations — typically used for lipophilic drugs — since they are characterized by a low viscosity and can be easily dispensed as eye drops. Consequently, in this section we will particularly concentrate in the variety of CS-based delivery vehicles for lipophilic molecules. sections, CS-based nanocarriers have been specifically designed to achieve improved encapsulation efficiencies for hydrophobic drugs and nucleic acids. Moreover, studies have been conducted to explore the biodistribution, biocompatibility and eventual efficiency of the developed CS nanocarriers (see Table 2). Overall, there is a certain evidence of the potential of CS for topical ocular administration. However, to the best of our knowledge, very limited experience exists with regard to its use in intraocular delivery. CS in solution has been recently proposed for the treatment of vitreous retinopathy, as it may inhibit fibroblastic proliferation without altering the inner structures of the eye or produce severe inflammatory responses [95]. Apart from this, DNA-loaded CS nanoparticles have unsuccessfully been employed for the transfection of the retina and retinal pigment epithelium [96]. 4. Chitosan-based delivery vehicles for small molecules Currently, there is a variety of nanostructures described in the literature with a capacity to entrap a wide range of molecules and Table 2 A brief presentation of different CS nanostructures, which have been investigated with regard to their ability to deliver different active compounds to the ocular surface as well as their biodistribution, safety and efficacy. Biodistribution Biocompatibility and toxicity CS-based nanoemulsions Lipophilic (CS nanocapsules) molecules [3,27] Biomolecules – CS-coated nanosystems CS-coated nanoparticles are able to effectively interact with the ocular mucosa, being this attributed to the intrinsic properties of CS [28] The association of indomethacin to CS nanocapsules led to a significant increase of the indomethacin concentration in the cornea and aqueous humour after topical instillation to rabbits [3] The association of indomethacin to CS-coated PECL nanocapsules exhibited CS-coated PECL nanocapsules led to a a good ocular tolerance following topical significant increase in the indomethacin ocular administration to rabbits[27,28] concentration in the cornea and aqueous humour after topical instillation to rabbits [27,28] The association of the immunosuppressive agent — rapamycin — to CS-coated PLA nanoparticles, led to a significant increase in corneal allograft survival in rabbits [109] The association of timolol to CS-coated niosomes led to an efficient and long-lasting control of the intraocular pressure [108] The association of CyA to CS nanoparticles CS nanoparticles exhibited low toxicity led to a significant increase in the CyA following incubation with cells derived concentration in the cornea [6] from the conjunctival epithelium [5,187]. They also showed adequate The association of pDNA to CS nanoparticles biocompatibility after acute administration led to the expression of the encoded protein to rabbits [187] upon their incubation with corneal and conjunctival epithelial cells [38] Lipophilic molecules [27,28,108,109] CS-coated-PECL nanocapsules are able to penetrate the cornea by a transcellular pathway [29] CS nanoparticles Self-assembled CS nanoparticles CS–polysaccharide nanoparticles CS–lipid nanoparticles CS–cholesterol-coated PLA nanoparticles show a good retention onto the eye surface [109] Lipophilic CS nanoparticles show an improved drugs [6] interaction with the ocular mucosa Proteins [7,187] (cornea and conjunctiva) with respect Nucleic acids [38] to the polymer in solution. They penetrate the corneal epithelium by a transcellular pathway and have a specific affinity for some conjunctival cells [5]. CS nanoparticles are actively taken up by conjunctival cells. The endocytic process is temperature dependent, but not energy dependent [187] Lipophilic drugs Cholesterol–CS nanoparticles show an evenly distribution and good [111,112] retention time after instillation onto the ocular surface [112] Hydrophilic HA/CS nanoparticles can interact molecules [176] with the HA-receptor CD44, expressed in ocular cell lines [38] Lipophilic HA/CS nanoparticles interact in molecules [35] Proteins [35] a big extent with the ocular mucosa after topical instillation Nucleic acids to rabbits [163] [38,163] Lipophilic molecules [113] Proteins [178] Liposome–CS nanocomplexes exhibited a specific interaction with the conjunctiva after topical administration [178] Efficacy in vitro/in vivo CS-coated nanoemulsions —also named as nanocapsules— exhibited a good ocular tolerance following topical ocular administration to rabbits [27] – HA/CS nanoparticles exhibited a very low toxicity (lower than that of CS nanoparticles) upon incubation with cell lines derived from the human cornea and conjunctiva [38] HA/CS nanoparticles did not induce alterations in the ocular function or cause any signs of irritation upon acute administration to rabbits [191] HA/CS nanoparticles were bioassimilated by the corneal cells [163] Liposome–CS nanocomplexes are well tolerated upon topical administration to rabbits [178] The association of prednisolone to CS nanoparticles led to a significant increase in the predinosolone concentration in the aqueous humour [111]. The association of pDNA to HA/CS nanoparticles led to a significant protein expression upon their incubation with corneal and conjunctival epithelial cells [38,163] The association of pDNA to HA/CS nanoparticles led to a significant and prolonged protein expression in the cornea, upon topical instillation to rabbits [163] – M. de la Fuente et al. / Advanced Drug Delivery Reviews 62 (2010) 100–117 4.1. Chitosan-based nanoemulsions and nanocapsules In the last decade, oil-in-water (o/w) lipid emulsions have been investigated as a vehicle intended to improve the ocular bioavailability of lipophilic drugs [20,98]. The o/w lipid emulsions can be easily classified into two types, anionic and cationic emulsions, on the bases of the surface charge impaired by the emulsifiers and/or lipids used for their preparation. Negatively charged emulsions are prepared using anionic lipids and surfactants, whereas positively charged emulsions can be prepared using cationic lipids, such as stearylamine and oleylamine [99,100]; alternatively, cationic polysaccharides, such as CS, can be used to form a coating around the oily droplets, thus impairing a positive charge to the emulsion, as will be discussed later. A number of anionic lipid emulsions have already been commercialized, as indicated in Table 1, Section 2, especially for the treatment of the dry eye syndrome. Comparatively, cationic emulsions appear to exhibit some additional advantages. For example, they facilitate the spreading onto the ocular surface and the establishment of electrostatic interactions with the ocular tissues. These properties lead to a consequent improvement of the residence time, an increased wettability of the ocular surface, and an improved capacity for delivering drugs [101]. The superiority of cationic submicron emulsions vs. anionic amulsions has been illustrated in the case of cyclosporin A [102,103]. Similarly, other authors have described the potential of cationic submicron emulsions for the ocular application of the anti-inflammatory drug piroxicam [100]. Concretely, they observed that the piroxicam-loaded positively charged emulsion reduced the ulcers induced in the rabbit cornea. The authors suggest the increased uptake of the positive oil droplets as a plausible explanation for the resulting enhancement of the lipophilic drug ocular disposition. All together, the cationic character of the emulsion and the subsequent interaction with the ocular surface appears to be the main factor governing the increase in the bioavailability of the encapsulated drug upon topical administration. Some authors have suggested the potential of cationic emulsions thanks to the use of cationic polysaccharides, i.e. CS. This polymer has proved to be a useful emulsifier that stabilizes emulsions and prevents coalescence by steric and electrostatic hindrance, without the help of any additional surfactant [104]. Apart from that, CS has shown stabilizing properties following its addition to poloxamer as well as to lecithin-stabilized lipid emulsions [105], thus forming CS-coated emulsions otherwise called nanocapsules — according to the general definition disclosed in the next section. CS nanocapsules were described for the first time by Calvo et al. [27]. They were easily obtained by a solvent-displacement technique, using a negatively charged phospholipid in order to facilitate the attachment of CS onto the surface of the nanoemulsion. The cationic polysaccharide was incorporated into the external aqueous phase in which the formation of the colloidal structures takes place. Some lipophilic drugs, i.e. indomethacin and diazepam, have been successfully incorporated into the formulation, with encapsulation efficiencies as high as 90% [27,106]. The results obtained following topical administration to rabbits are particularly attractive as CS nanocapsules were shown to provide a significant increase in the corneal penetration of indomethacin [3]. Indeed, at earlier time points (1 h post-administration), the levels of indomethacin in the cornea and aqueous humour were found to be 30 times higher when encapsulated into CS nanocapsules, with respect to those achieved after instillation of a solution of indomethacin. Moreover, CS nanocapsules maintained a therapeutic concentration of indomethacin for at least 6 h post-instillation, in contrast to the 4 h observed for the indomethacin in solution. The authors attributed the obtained increase in the penetration and duration of indomethacin in the ocular structures as due to the mucoadhesive and penetration-enhancing properties of CS. 107 Similarly, CS has been employed as a coating material for poly-εcaprolactone (PECL) nanocapsules [27,106]. These and other CS-coated nanosystems are disclosed in the next section. 4.2. Chitosan coated nanocapsules and nanoparticles Nanocapsules are normally defined as colloidal carriers composed of an oily or an aqueous core surrounded by a polymer coating/shell. The first evidence of the efficacy of nanocapsules for ocular topical delivery of lipophilic drugs dates from 1992, when Losa et al. [22] compared the performance of poly-ε-caprolactone (PECL) nanocapsules containing metipranolol with the commercial eye drops. These polyester-based nanocapsules were able to reduce the intraocular pressure (IOP), while minimizing the cardiovascular side effects observed for the commercial eye drops. A similar decrease of IOP was obtained by Marchal-Heussler et al. [107] upon encapsulation of the antiglaucomatous drug carteolol into PECL nanocapsules. In the same line of research, the studies performed by Calvo et al. [25] showed that the inclusion of indomethacin into PECL nanocapsules led to an important increase in the penetration rate of the drug across the cornea. Moreover, the mechanistic studies performed by the same authors revealed that the colloidal nature of nanocapsules was a critical parameter responsible for the increased corneal penetration of indomethacin. Taking into account the improved drug transport obtained with these polyester delivery vehicles, in 1997 Calvo et al. developed a new type of cationic nanocapsules, which incorporated the mucoadhesive polymer CS [106]. The rationale for designing these CS-coated nanocapsules was to combine the advantages of PECL nanocapsules as ocular drug carriers with the mucoadhesive and permeability enhancing properties of CS. The efficacy of these new carriers was assayed for the anti-inflammatory drug indomethacin. The results showed that indomethacin-loaded CS-coated PECL nanocapsules provided an enhanced drug penetration to the cornea and aqueous humour, measured at 30, 60, 120 and 240 min post-instillation, as compared to the uncoated nanosystems or the commercial drug preparation [28]. In the same study it was shown that the positive response observed for these nanocapsules could not be simply attributed to their positive charge but to the inherent nature of CS. As a matter of fact, the comparative in vivo evaluation of indomethacin-loaded PECL nanocapsules coated with two cationic polymers, CS and poly-L-lysine, led to very different results. Namely, CS-coated PECL nanocapsules were able to significantly enhance the corneal penetration of indomethacin whereas the poly-L-lysine-coated nanostructures failed to provide any significant effect [28]. The superiority of CS as compared to other polymer coating materials, i.e. carbopol, was also shown in the case of timolol maleate [108]. More in detail, the pharmacodynamic evaluation of CS-coated niosomes containing timolol showed a peak effect at 3 h post-administration and an ability to control the IOP for 8 h after instillation onto the eye surface. In contrast, in the cases of carbopol-coated niosomes and the commercial formulation, they showed a peak effect at 3 h and 2 h post-instillation, respectively, and a shorter response, as it was maintained for up to 6 h and 5 h respectively. PLA hydrophobic nanoparticles have also been successfully coated with CS thanks to the use of an amphiphilic derivative of CS: cholesterol-modified CS (CS–CH) [109]. The resulting nanoparticles were used for the delivery of the hydrophobic immunosuppressive agent rapamycin (RAPA). The results showed that the positively charged coating endowed the nanoparticles with good retention ability at the precorneal area, thus facilitating the sustained release of RAPA to the corneal epithelium. Moreover, the efficacy of RAPAloaded CS–CH/PLA nanoparticles was investigated for the treatment of corneal allografts in rabbits. For that purpose, animals were treated with the RAPA-loaded nanoparticle suspension or the RAPA suspension twice a day during four weeks following surgery. The results 108 M. de la Fuente et al. / Advanced Drug Delivery Reviews 62 (2010) 100–117 evidenced an excellent immunosuppressive effect of the RAPA-loaded nanoparticles compared to the eye drops, as noted by the significant increase of the allograft median survival time of rabbit bearing corneal allografts [109]. 4.3. Chitosan nanoparticles Considering the success of CS nanoemulsions and CS-coated nanosystems for the delivery of lipophilic drugs to the eye, in 1997 Calvo et al. took up the challenge of designing a new type of nanosystem composed by CS as the main component [34]. CS is a cationic polysaccharide able to gel when in contact with specific multivalent polyanions, such as sodium tripolyphosphate (TPP). Nanoparticles are spontaneously formed upon mixing of CS and TPP solutions, through the formation of inter- and intramolecular linkages between the phosphate groups of TPP and the amino groups of CS [34]. Using this technique, it has been possible to efficiently associate hydrophilic compounds such as small molecules, peptides, proteins and genes. The establishment of electrostatic interactions either with the positively charged polymer CS or with the negative polyanion TPP is the main mechanism that governs the entrapment of these active compounds [7]. Despite of the hydrophilic nature of CS nanoparticles, it has been possible to entrap within their structure hydrophobic molecules by introducing some modifications in the nanoparticles preparation technique. In more detail, the entrapment of the hydrophobic polypeptide CyA, was achieved by a previous dissolution of the peptide in an acetonitrile:water mixture, and a further nanoprecipitation into the nanoparticles in the form of small nanocrystals. Entrapment efficiencies were reported to be as high as 73.4% [6]. The in vivo evaluation of this new prototype, in rabbits, evidenced the capacity of these nanoparticles providing a selective and prolonged delivery of CyA to the cornea and conjunctiva, as shown in the Fig. 3. In this figure it can be noted that the maximum levels of CyA, in both the cornea and the conjunctiva, were reached at 2 h post-instillation, the first time point under evaluation, and decreased gradually over time. More importantly, it was observed that CyA-loaded CS nanoparticles provided therapeutic levels of CyA in the conjunctiva and the cornea for up to 24 and 48 h postadministration, respectively, while reducing the access of CyA to the blood circulation. This positive behavior of CS nanoparticles was attributed to their improved interaction with the corneal and conjunctival and the prolonged delivery of the CyA molecules associated to them. Using a very similar technological approach other authors have associated other lipophilic molecules to CS nanoparticles. This is the case of indomethacin whose entrapment into CS nanoparticles was achieved by a previous dissolution in methylene chloride and a further nanoprecipitation into the CS nanoparticles [3]. Unfortunately, no evidence of the in vivo behavior of this formulation has been reported so far. Another strategy for the encapsulation of lipophilic compounds within CS nanostructures has involved the chemical modification of the polysaccharide with hydrophobic residues. The resulting polymers have exhibited an ability to self-assemble as supramolecular nanostructures due to their amphiphilic character. For example, quaternary ammonium palmitoyl glycol CS aggregated into a hierarchically organized micellar cluster enable the entrapment of hydrophobic compounds, such as prednisolone [110,111]. Following topical ocular administration to rabbits, it was found that these new nanostructures have a capacity for overcoming the ocular barriers. Concretely, the levels of prednisolone achieved in the aqueous humour following administration of the nanostructures were comparable to those obtained after administration of a ten-times more concentrated commercial suspension of the drug. In a different study, the formation of self-aggregated nanoparticles consisting of amphiphilic conjugates of CS and cholesterol (CH) was Fig. 3. Concentration of [3H]-CyA in A) the cornea and B) the conjunctiva after topical administration to rabbits of CyA-loaded CS NPs (black columns), a suspension of CyA in a CS solution (stripped columns) or a suspension of CyA in water (white columns). The CyA levels were determined by liquid scintillation at 2, 6, 24 and 48 h after instillation. Statistical differences are denoted as *p b 0.05. Adapted from [6] with permission. described (CS–CH) [112]. The ocular distribution of radiolabelled CS–CH aggregates, upon instillation onto the ocular surface in rabbits, was assessed by scintillation counter and single photon emission computed tomography. Results showed a good spreading of the nanoparticles over the precorneal area and a residence time of 2 h. 4.4. Chitosan/cyclodextrin nanoparticles Despite of the success of CS nanoparticles for the association and delivery of lipophilic molecules, a number of studies have been recently aimed at modifying their composition in order to increase their versatility and minimize their toxicity [36,113]. Based on these considerations, Maestrelli et al. developed a new nanoparticulate drug carrier, which combines the benefits of CS and cyclodextrins [36]. Cyclodextrins are a family of cyclic oligosaccharides which form M. de la Fuente et al. / Advanced Drug Delivery Reviews 62 (2010) 100–117 inclusion complexes with hydrophobic molecules leading to an improvement of their solubility and/or stability [114,115]. These novel nanocarriers combining different cyclodextrins and CS have been found to be adequate carriers for the association of hydrophobic molecules as well as hydrophilic proteins [116] and genes [117]. In addition, they have shown excellent properties for transmucosal delivery of macromolecules across the nasal mucosal route [116]. However, they still need to be evaluated with regard to their potential for ocular drug delivery. Given the previous evidence of the potential of CS-based nanostructures as well as that of drug–cyclodextrin inclusion complexes [118], it could be presumed that the combination of them would provide specific benefits as ocular drug delivery systems. 5. Chitosan-based delivery vehicles for gene therapies Over the few last years, researchers have identified several nucleic acid-based therapies which may be of benefit in treating different disorders of the eye, thus representing an increasingly important segment of the therapeutic arsenal in ophthalmology [13,93,119– 121]. These new therapies offer important advantages with respect to conventional treatments, especially in terms of selectivity. The transfection of the ocular mucosa may be of interest, not only for the local treatment of the corneal and/or conjunctival epithelium but also for the treatment of other pathologies which affect the inner eye [122]. However, as mentioned in Section 2.2, the potential of gene therapy is compromised by a significant obstacle: the delivery of these drugs to the target site [10,42,52,92]. Numerous synthetic carriers have been proposed in the last few years to achieve this goal [17]. Overall, several critical steps have to be considered for designing an optimal synthetic vector for topical ocular gene delivery. First at all, gene nanocarriers should be able to entrap the nucleic acids and efficiently deliver them to the ocular cells. Secondary, they must interact with the ocular mucosa and transfect the ocular epithelia under physiological conditions, leading to the desired therapeutic effect. 5.1. Chitosan–DNA complexes Most commonly, non-viral nanocarriers are based on the establishment of electrostatic interactions between the negative charged nucleic acids and the positive charged polymers (polyplexes), lipids (lipoplexes) or dendrimers (dendriplexes). Fairly known cationic polymers, such as polyethyleneimine (PEI), as well as cationic lipids and dendrimers, have already been described for their specific application in topical ocular gene therapy, showing promising results both in vitro and in vivo [123–125]. However, to the best of our knowledge, little information is available concerning the toxicological profile of these compounds in ocular cells. Lipoplexes have demonstrated limited toxicity after incubation over corneal endothelial cells. Nonetheless, differences were detected such as being dependent on the amount of lipids and the lipidic composition [125]. CS is another cationic polymer with a potential in ocular gene therapy. Apart from the specific claims that make it an interesting and unique polymer for ocular drug delivery (see Section 3), CS has been widely explored with respect to its ability to complex nucleic acids and act as an effective gene carrier [126–129]. Some key properties of the polysaccharide, for example its molecular size, deacetylation degree and stechiometry of the complexes, have been systemically investigated with respect to their influence on the transfection ability of the CS nanocomplexes. Among them, the CS molecular weight was found to significantly affect its ability to transfect mammalian cells. As reported in several studies, a low molecular weight strongly favours the process of gene transfection [128,130–136]. The success of CS-based nanocomplexes for the delivery of nucleic acids to mucosal surfaces has been shown in vivo after pulmonary 109 administration [137,138]. With respect to their application for the delivery of interferents, CS nanocomplexes loaded with siRNA were able to effectively interfere in the expression of the target model protein EGFP (enhanced green fluorescent protein), following intranasal administration to transgenic mice [139]. Besides these promising results, one of the major disadvantages of CS nanocomplexes, and polyplexes in general, is related to the simplicity of their structure. Since plasmid and polymer are held together by the simple establishment of electrostatic interactions, the presence of polyanions naturally present in the body, such as heparin and glycosaminoglycans, may lead to the unpacking and release of the pDNA, before reaching the target site [140–143]. This limitation could perhaps explain the lack of information regarding the use of CS complexes for ocular gene delivery. In this sense, the development of more stable delivery systems, in the form of nanoparticles, could be understood as a way of improving the potential of CS in ocular gene therapy. 5.2. Chitosan nanoparticles CS nanoparticles produced by ionotropric gelation are considered to be adequate nanocarriers for the encapsulation of labile nucleic acids. As indicated in a previous section (Section 4.2), the mechanism that governs the formation of the nanoparticles is the controlled gelation of CS due to its interaction with the phosphate groups of the crosslinking agent sodium tripolyphosphate [34]. This controlled gelation allows the formation of round and homogeneous nanoparticles, regardless of the polysaccharide molecular weight [144,145], considered to be an important parameter in gene delivery applications. Our research group and others have reported the utility of CS nanoparticles as delivery systems for DNA [37,143], oligonucleotides (ODN) [146], and RNA of interference (i.e. siRNA and shRNA) [147,148]. These nanocarriers have specific advantages, compared to the simple CS nanocomplexes, such as a spherical and homogeneous shape, an improved stability (DNA molecules are not displaced by other anions) and controlled release of the associated DNA [143]. These advantages have also been evidenced for siRNA molecules [147]. Finally, an additional benefit of CS nanoparticles is that their physicochemical properties, such as size and zeta potential, which are important properties that determine the gene transfection efficiency, can be easily controllable by the adequate selection of the processing parameters, such as the CS to TPP ratio [145]. The success of CS nanoparticles for the delivery of plasmid DNA to mucosal surfaces such as the oral and the nasal mucosa has already been shown [149–151]. With respect to their specific application as ocular gene carriers, there is evidence of their ability to transfect ocular cells in vitro, more specifically cell lines derived from the human cornea and the conjunctiva, respectively Human Corneal Epithelial (HCE) and Normal Human Conjunctival (NHC-IOBA) cells. This capacity of CS nanoparticles to transfect the cells, was found to be highly dependent on the molecular weight of CS. In fact, only CS of low molecular weight, i.e. 10–12 kDa, was able to induce the expression of the reporter protein EGFP in both cell lines [38]. Additionally, in these studies it was observed that the capacity of CS nanoparticles to transfect ocular cell lines could be significantly increased by addition of a second ingredient to the nanoparticle composition, i.e. hyaluronic acid, as disclosed in the next section (Section 5.3). Hence, the optimization of CS-based nanoparticles as ocular gene carriers may start by the rational selection of new materials which would lead to an increase on the capacity of CS-based nanoparticles to transfect ocular tissues. 5.3. Chitosan/hyaluronic acid nanoparticles A particularly interesting formulation for the ocular delivery of nucleic acids, which has been reported by our group, consists of a 110 M. de la Fuente et al. / Advanced Drug Delivery Reviews 62 (2010) 100–117 hybrid nanostructure of CS and the natural occurring glycosaminoglycan hyaluronic acid (HA) [35]. The selection of this anionic polysaccharide was based on its interesting properties, i.e. mucoadhesiveness, biocompatibility and biodegradability [152–155] and also on its ability to selectively interact with the ocular mucosa. In fact, it is well known that HA may act as a targeting ligand to the CD44 receptor [123,156–158], which is expressed in the ocular epithelia [159–161]. The surface modification of nanoparticles with specific ligands may be a promising strategy to increase the nanoparticle uptake and transport. Indeed, Kompella et al. [162] have demonstrated that the cellular uptake of transferrin or desorelin-modified polystyrene nanoparticles, able to specifically interact with transferring and LHRH receptors, was significantly improved when compared with plain nanoparticles. With regard to the formulation developed in our group, the composition of HA/CS nanoparticles could be easily modulated by adjusting the formulation conditions (HA and CS amounts and molecular weight, as well as the amount of the crosslinker tripolyphosphate) [35]. In addition, we have reported their great capacity for the association of DNA (pDNA) and RNA of interference (RNAi), while preserving their structure and biological activity [163–165]. The ability of HA/CS nanoparticles to transfer genes (pDNA) to ocular cells was first screened in vitro [38] and the best prototypes, according to their ability to transfect ocular epithelial cell lines were later selected for further in vivo evaluation [163]. HA/CS nanoparticles loaded with the model plasmid EGFP, which encodes a fluorescent protein and allows an easy determination of the level of gene expression, were used for the transfection experiments performed in HCE and NHC-IOBA cell lines [38]. In this particular work it was shown that the proportion of HA in the nanoparticles influenced positively the levels of detected protein. This effect was not casual but expected given the positive influence of the glycosaminoglycan with respect to the efficiency of gene transfer [166,167]. In addition, this positive effect of HA was attributed to the capacity of these nanocarriers to specifically interact with the hyaluronan receptor CD44. As a matter of fact, when the receptor was blocked with a monoclonal anti-CD44 antibody or with an excess of free HA, a significant decrease in the amount of protein expressed was observed. The molecular weight of CS also showed a strong influence with respect to the ability of the HA/ CS nanoparticles to transfect the ocular cell lines: higher levels of protein expression were observed when the nanoparticles were composed by hyaluronic acid (HA) and oligomers of CS (CSO); i.e. HA/ CSO nanoparticles, in agreement to what has been observed for CS complexes and nanoparticles (see Sections 5.1 and 5.2). A model of the corneal epithelium was eventually used for evaluation of the capacity of HA/CS nanoparticles to transfect differentiated human corneal epithelial cells (HCE-model) [163]. Indeed, the transfection of differentiated corneal cells that closely resemble the ocular epithelium, HCE-model [122,168,169], was expected to provide more accurate information and confirm the selection of the best prototypes for a final in vivo evaluation. As expected, HA/CSO nanoparticles provided very encouraging results in their role as gene carriers for topical ocular gene delivery. Finally, in order to obtain a proof-of-concept of the ability of HA/CS nanoparticles to act as gene carriers in vivo, HA/CSO nanoparticles were loaded with a plasmid encoding EGFP protein and subsequently instilled in rabbits. The kinetics of gene expression was evaluated under a confocal microscope [163]. Interestingly, the results showed a long-term expression of up to 1 week of the encoded green fluorescent protein in the corneal epithelium. Images of the rabbit cornea expressing the green fluorescent protein can be observed in Fig. 4 [163]. Overall these results show the very positive behavior of HA/CS nanoparticles as ocular gene carriers and open up a promising expectative for the therapeutic application of nucleic acids in ophthalmology, not only for the treatment of disorders that affect the ocular surface but also the internal structures of the eye. In this sense, it is important to mention a report in which it has been shown how the transfected cornea could actually behave as an efficient platform for the prolonged delivery of secreted proteins into both the lachrymal fluid and the aqueous humour [122]. 5.4. Chitosan/alginate nanoparticles Alginate (ALG) is another interesting polysaccharide which has been previously used in the preparation of several ocular delivery systems and in combination with other materials, such as ALG/ hydroxypropyl methylcellulose hydrogels [170], ALG/collagen microspheres [171], or ALG/chitosan films [172]. The formation of chitosan/ alginate nanoparticles (CS/ALG nanoparticles), by the ionic gelation technique, has been recently disclosed [173–175]. The resulting nanoparticles have shown a capacity to effectively entrap and protect biomolecules of therapeutic interest. With respect to their use in ophthalmology, CS/ALG nanoparticles have been specifically designed for the prolonged delivery of the antibiotic gatifloxacin to the ocular mucosa [176]. On the other hand, it has been reported that the incorporation of ALG is an efficient strategy for increasing the transfection efficiency of CS nanoparticles, as it modulates and improves the delivery of the associated plasmid [177]. However, despite the theoretical interest of these nanoparticles for gene delivery to the eye, their application by this particular route remains to be explored. 5.5. Chitosan–lipid complexes It has been shown that the incorporation of different biomaterials into CS nanostructures may lead to the development of interesting carriers with new and improved properties. Among them, our group has embraced the idea of generating a new type of nanosystems that combine the positive features of polysaccharides with those described for lipids. The rational mixture of CS with phospholipids or with preformed liposomes has led to the formation of supramolecular hybrid structures [113,178]. These nanostructures have demonstrated an adequate stability in biological fluids and are suitable for the encapsulation of labile macromolecules, such as insulin [179]. An Fig. 4. Confocal images of the transfected corneal epithelium that express the reporter green fluorescent protein. pEGFP-loaded HA/CSO NPs were topically instilled onto the eye surface of rabbits (50 µg pDNA/cornea) and the efficacy and duration of the gene expression evaluated at 2, 4 or 7 days after administration (n = 3 specimens). Adapted from [163] with permission. M. de la Fuente et al. / Advanced Drug Delivery Reviews 62 (2010) 100–117 additional property is their ability to control the release of the entrapped molecules as a function of the lipidic composition [180,181]. With respect to their specific application in ophthalmology, it has been shown that CS–lipid nanocomplexes can efficiently interact with ocular tissues and enter the ocular cells, without compromising the integrity of the cellular membrane [178]. While the efficacy of these nanosystems as gene carriers remains to be proven, the results reported until now suggest their potential as ocular gene delivery systems. On the one hand, CS nanoparticles were able to transfect both corneal and conjunctival cells, and it has been shown that the inclusion of a second biomaterial could significantly increase their efficacy as gene carriers [38]. On the other hand, liposomes are considered to be efficient systems for transfection; actually, some lipidic compositions have already shown a certain degree of success in ocular gene therapy [125,182]. 6. Biodistribution, toxicity and biocompatibility 6.1. Biodistribution: interaction of Chitosan-based nanoparticles with the ocular epithelia CS has been widely described as a highly mucoadhesive polysaccharide able to establish an intimate interaction with the mucus layer that covers the ocular mucosa. In fact, and as discussed earlier (Section 3) the spreading use of CS in ophthalmology has a basis on these properties, as it has been proven that CS can increase the residence time of the administered drug while promoting its permeability through the ocular epithelium [70,82,173,183–186]. The ability of CS to effectively interact with the ocular mucosa has been attributed not only to its cationic charge but also to its intrinsic properties [28]. In addition, we have precisely shown that the presentation of CS in a nanoparticulated form significantly increases its retention at the ocular surface and, thus its utility for ocular drug delivery [5]. With the aim of evaluating the influence of the CS presentation on the interaction of nanostructured systems with the ocular mucosa, we have compared the intensity and pattern of interaction of two different systems, CS nanoparticles and CS-coated PECL nanocapsules, using both fluorimetry and confocal microscopy. As indicated before, CS-coated PECL nanocapsules are composed of a lipid core, which is surrounded by a PECL wall and a fluid CS coating. In contrast, CS nanoparticles are solid nanomatrices consisting of crosslinked CS. CS-coated PECL nanocapsules, loaded with the fluorescent tracker rhodamine B, were incubated with the excised rabbit cornea ex-vivo and, then, imaged at the confocal microscope [29]. The images taken allowed us to visualize fluorescent spots uniformly distributed inside the cells that clearly indicated that nanocapsules were able to penetrate into the corneal epithelium by a transcellular pathway. In order to corroborate these results, a second set of experiments was performed in vivo, for which the nanoparticles were directly instilled onto the eye surface of conscious rabbits. The biodistribution pattern in the corneal epithelium was identical to that observed in the ex-vivo experiment, thus reaffirming the observation that CS-coated nanocapsules follow a transcellular route. The significance of the surface composition of the nanosystems was also established in the same work, as it was observed that both the extent of the interaction and the deep of penetration through the corneal epithelium are facts directly related to this parameter. For example, systems coated with CS exhibited an important interaction but a low penetration depth into the corneal epithelium, compared to those coated with PEG. In addition, this important interaction led to a more significant increase in the transport of the tracker rhodamine B when encapsulated into CS-coated systems. This fact may also explain previous findings, i.e. the increased levels of indomethacin detected both in the cornea and intraocular structures when encapsulated into CS-coated nanocapsules, versus both uncoated and poly-L-lysine-coated nanocapsules [28]. 111 The interaction of CS nanoparticles with the ocular mucosa has been explored in a similar way to what described for CS-coated nanocapsules. For that purpose, a fluorescent tag was chemically bound to the polysaccharidic molecule, in a previous step to the preparation of the nanoparticles. The nanoparticle suspension, containing both nanoparticles and soluble CS, was instilled onto the eye surface of rabbits [5]. Surprisingly, the CS distribution was found to be different to what previously observed for CS-coated nanosystems. The pattern of fluorescence situated the labelled CS at the boundary region between corneal epithelial cells and, to a lower extent, in the intracellular space. This observation led to the conclusion that the labelled CS was able to enter the epithelia by a combined paracellular/transcellular pathway. The transcellular pathway is in accordance with what was previously described for CS-coated nanoparticles. With respect to the paracellular transport, it was attributed to the presence of soluble CS molecules in the formulation, which may open the tight junctions and act as a permeabilization enhancer. Another observation extracted from this study refers to the different patterns of distribution found in the corneal and conjunctival epithelium. In fact, while the fluorescent signal was uniformly distributed in the cornea, an uneven distribution was observed in the conjunctiva. As the conjunctiva is a heterogeneous epithelium, the different localization of the nanoparticles could be explained by a greater affinity of the nanoparticles by some specific type of cells. Moreover, the intensity of the fluorescence in the conjunctiva was higher as compared to the cornea, which was justified by considering the more important concentration of mucin in the conjunctiva accompanied by a greater chance of the mucoadhesive CS nanoparticles to adhere to it [5]. This pattern of interaction of CS nanoparticles with the cornea and the conjunctiva was further confirmed after topical instillation of fluorescent albumin (FITC-BSA) loaded CS nanoparticles to rabbits [187]. In addition, they were performed in vitro mechanistic studies concerning the interaction among CS nanoparticles and conjunctival epithelial cells, using for that purpose an immortalized cell line derived from the conjunctival epithelia (NHCIOBA). The confocal images of NHC-IOBA cells exposed to FITC-BSAlabelled nanoparticles showed a great number of uniformly distributed fluorescent spots inside the cells, thus evidencing the capacity of CS nanoparticles to be actively taken up by conjunctival cells. The mechanism of endocytosis was found to be temperature dependent, but no metabolic energy seemed to be required. Similar results were obtained when the biodistribution of lipid– chitosan nanoparticles (LCS-nanoparticles) was investigated [178]. In this case, and for visualization of the LCS-nanoparticles in the confocal microscope, FITC-BSA was associated to the nanostructure. Results evidenced a specific affinity of the complexes for the conjunctival epithelium. Even if detectable fluorescence signals corresponding to the FITC-BSA associated to the complexes were observed in the basolateral membranes and throughout the cytoplasm of the corneal cells, these signals were lower than those observed in the conjunctival epithelium. Interestingly, the fluorescent pattern observed in the conjunctiva was strongly dependent on the lipidic composition of the LCS-nanoparticles. Studies performed in vitro, in a multilayered, mucus-like producing primary culture of the conjunctival epithelium, reached to the same result. The complexes were found to interact with the mucus layer to different extents depending on the formulation. In fact, their internalization was apparently delayed by the presence of mucus to a certain degree strictly dependent on the composition of the liposomes. In Fig. 5 it can be clearly seen that the intensity of the fluorescence corresponding to the labelled LCS-nanoparticles (green signal) after deposition over primary cultures of the conjunctiva (red signal), strongly varies for the different formulations as a function of the lipidic composition. Hence, the importance of the lipidic composition on the mucoadhesion of the complexes was the most relevant information extracted from this study. This finding may be of 112 M. de la Fuente et al. / Advanced Drug Delivery Reviews 62 (2010) 100–117 Fig. 5. Confocal images that show as liposome:chitosan nanoparticles complexes (2:1), LCS-NP (green), interact in a different degree with a mucus-like producing primary culture of the human conjunctival epithelium (red) as a function of the lipidic composition (LCS-NP1, LCS-NP2 and LCS-NP3), and after 30 min incubation (n = 3 specimens). The liposome formulation of LCS-NP1 consisted of DSPC:DPPS:Chol (6:0.1:4 molar ratio), LCS-NP2 of DPPC:Chol (6:4 molar ratio), and LCS-NP3 of DSPC:Chol (6:4 molar ratio) (DSPC: Distearoylphosphatidylcholine. DPPS: Dipalmitoylphosphatidylserine. Chol: Cholesterol). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Adapted from [178] with permission. great interest in the design of specific carriers capable of modulating the retention time of a particular encapsulated drug at the conjunctival epithelium. However, more mechanistic studies need to be carried out in order to fully understand the main factors affecting the affinity of these nanocarriers for the conjunctival epithelia and therefore providing the necessary tools for the design of delivery systems with more defined properties. The retention of a different nanostructure consisting of nanoparticles made of cholesterol-modified chitosan (CS–CH nanoparticles) onto the ocular surface has also been explored [112]. In this case, CS was radiolabelled and the ocular distribution of the nanostructures imaged by single photon emission computed tomography (SPECT). The authors observed that the self-assembled CS–CH nanoparticles spread over the entire precorneal area immediately after the topical administration. Minutes later, part of the suspensions drained into lachrymal duct and, finally, into the lachrymal sac. However, a major percentage of radioactivity (72%) remained at the ocular surface after 2 h post-instillation, thus indicating the good retention of CS–CH nanoparticles in the precorneal area. In a different work, the interaction of solid poly-lactic acid (PLA) nanoparticles coated with CS–CH, with the ocular mucosa, was also investigated by applying the same technology (SPECT) [112]. Overall, and besides the common nature of CS–CH, a different behavior can be observed with both types of nanostructures, self-assembled CS–CH nanoparticles and PLA-coated CS–CH nanoparticles [109,112]. The results indicated that, contrary to the behavior of self-assembled CS–CH nanoparticles, which spread well onto the whole ocular surface, these CS–CH-coated PLA nanoparticles aggregated and remained in the conjunctival sac, this fact being attributed to the high viscosity of the CS solution in the nanoparticles preparation. While considering the increased hydrophobic character of the PLA-coated CS–CH nanoparticles, it should also be mentioned that similar patterns of distribution have been previously observed for other highly hydrophobic nanostructures, such as PECL nanoparticles [107]. As indicated in a previous section (Section 5.3), we have recently explored a strategy for targeting nanoparticles to corneal and conjunctival cells. We chose HA as a targeting ligand to be incorporated into CS nanoparticles, given to its specific interaction with the CD44 receptor, which is expressed by the corneal and conjunctival epithelial cells [161,188,189], and to the positive implications that this interaction may have in the transfection efficiency of the nanoparticles [166,167,190]. As discussed, in order to corroborate the hypothesis of the specific interaction of HA/CS nanoparticles with the CD44 receptor, we first studied this mechanism using CD44-positive human corneal epithelial cells (HCE). The results showed that a specific interaction occurred and, moreover, that it has a direct implication on the success of these nanocarriers as gene vectors to ocular epithelial cells [38]. In a second step, we investigated the interaction of fluorescent HA/CS nanoparticles with the corneal and conjunctival epithelium following topical administration to rabbits. The results showed that HA/CS nanoparticles could enter the ocular mucosa in a very effective way. Even if no significant differences were visually appreciated, concerning the nanoparticles distribution in the cornea and the conjunctiva [163], upon quantitative determination the amount of fluorescence in the conjunctiva was found to be higher [191]. Another piece of relavant information concerns the different behavior that HA/CS nanoparticles show, with respect to the previously studied LCS-nanoparticles described above, as their internalization by the conjunctival epithelium was not delayed as a consequence of the mucoid component; the HA/CS nanoparticles seem to have overcome this limitation [191]. Overall, CS-based nanoparticles show good retention over the ocular surface after topical instillation, and are able to enter the corneal and the conjunctival epithelium by a transcellular pathway. CS-based nanoparticles show a limited penetration but a strong interaction with the ocular mucosa, all together leading to an improvement in the delivery and transport of the encapsulated molecules. Additionally, a specific preference of CS nanoparticles for the conjunctiva has been observed. Similarly, LCS-nanoparticles show a stronger interaction with the conjunctiva. In this case, the lipidic counterpart influences the general behavior of the nanosystems with respect to their interaction with the mucus layer that covers the conjunctival epithelium. HA/CS nanoparticles manifested an improved interaction with the ocular surface. In general, it appears that the interaction of the CS-based nanostructures with the superficial tissues of the eye (cornea and conjunctiva) may depend on their relative affinity for the mucus and the epithelial cells. However, more mechanistic studies need to be carried out in order to fully understand the mechanisms involved in this interaction. 6.2. Toxicity and biocompatibility Ocular biocompatibility and toxicity are critical issues to consider for the design of nanostructured ocular drug delivery systems; especially when considering that for various clinical applications drug delivery to the eye requires chronic administration. Therefore, the biodegradability of the nanostructures becomes a basic requirement in order to avoid their accumulation in the ocular tissues. Biocompatibility is a multidimensional concept, which has recently been redefined as follows [192]: “Biocompatibility refers to the ability of a biomaterial to perform its desired function with respect to a medical therapy, without eliciting any undesirable local or systemic M. de la Fuente et al. / Advanced Drug Delivery Reviews 62 (2010) 100–117 113 effects in the recipient or beneficiary of that therapy, but generating the most appropriate beneficial cellular or tissue response in that specific situation, and optimising the clinically relevant performance of that therapy”. For the specific application of topical delivery to the eye surface, several tests measuring the eye function, as well as signs of discomfort and irritation, have been specifically described (see Section 6.2.2). As mentioned before (Section 3) CS is known as a biodegradable and biocompatible polymer, suitable for delivery to mucosal routes. For example, early studies by Hirano et al [193] presented CS as a safe material for both oral and intravenous administration. Similarly, Felt et al. [31] identified CS as a well tolerated polymer adequate for the development of ophthalmic formulations intended to the topical route. With respect to the biocompatibility of nanoparticulate systems, much of the published data suggest that an appropriate particle size and a narrow size range ensure low irritation, adequate bioavailability and good compatibility with the ocular tissues [1,194]. Despite of this, a number of studies both in vitro and in vivo were aimed to assess the biocompatibility and tolerance of CS-based nanoparticles. 6.2.1. In vitro studies In vitro tests were performed for determining the toxicological profile of CS-based nanoparticles on immortalized cell lines obtained from the ocular epithelia, i.e. cornea (HCE) and conjunctiva (Chang and NHC-IOBA cells). Initial reports concerning the cytotoxicity of CS nanoparticles indicated acceptable toxicological profiles in vitro, as the levels of cell death did not exceed 5% for a polymer concentration of 2 mg/ml [5,187]. Similar results were obtained in the case of complexes of CS nanoparticles with liposomes (LCS-nanoparticles) [178]. In general, the cell viability after exposure to LCS-nanoparticles was higher than that observed for CS nanoparticles. The toxicity of CS nanoparticles and LCS-nanoparticles was found to be concentration dependent, the cell viability being higher for 0.25 and 0.5 mg/ml compared to 1 mg/ml. With respect to the incubation time, percentages of cell viability greater than 60% were found when the LCS-nanoparticles remained in contact with the cells for 30 min. In addition, the cytotoxicity of the complexes was evaluated in a primary culture derived from the conjunctiva, which closely resemble the conjunctival epithelium. The observation by confocal microscopy of the conjunctival primary cultures confirmed that no alteration in their morphology occurred after exposure to LCSnanoparticles for 30 min. Overall, the results indicate that LCSnanoparticles exhibit an adequate toxicological profile for administration onto the eye surface. Besides the low cytotoxicity of CS nanoparticles, a significant improvement on the toxicological profile was observed when HA was included into the nanostructure. This is logical since HA is a natural biomaterial involved in several processes such as cell migration and proliferation [195]. As a matter of fact, HA is widely used in ophthalmology, for example as a wound-healing agent for corneal wounds and injuries [196,197], as a substitutent of the vitreous and the aqueous humour [198] or for the preparation of artificial lachrymal fluid [199]. Our group has evaluated the cytotoxic effects in HCE and NHCIOBA cell lines, derived from the human cornea and conjunctiva, respectively, of HA/CS nanoparticles of different compositions (with respect to the proportion of both polysaccharides) [38]. Encouraging results show a gradual increase in the rate of cell survival as the amount of HA in the nanoparticles increases, reaching 100% of cell viability for nanoparticles composed by HA and CS in a proportion 2:1 (Fig. 6 for illustration). Therefore, highly biocompatible nanoparticles could be elaborated by simply adjusting the nanoparticles composition. 6.2.2. In vivo studies Early studies performed by our group, suggested that CS-coated nanocapsules are well tolerated nanocarriers that do not cause microscopically visible changes in the morphology of the corneal Fig. 6. Cytotoxicity curves obtained after 1 h exposure of A) corneal (HCE) or B) conjunctival (NHC-IOBA) epithelial cells to pEGFP-loaded CS (-×-) or pEGFP-loaded HA/CS NPs in a proportion HA:CS of 1:2 (-●-), 1:1 (-■-) or 2:1 (-▲-). The cell viability was measured by applying the MTS assay (means ± S.D., n = 3). Statistical differences are denoted as *p b 0.05. Adapted from [38] with permission. epithelium [28]. Some years later we evaluated the topical tolerance of CS nanoparticles, following their topical administration to rabbits [5,187]. Namely, the nanoparticles were administered to rabbits every 30 min for 6 h, at a concentration of 0.5 mg/ml, and the eyes were observed for any possible sign which could denote a lack of tolerance or the existence of inflammatory or dysfunction processes (for example an increase in the frequency of blinking, opacification of the cornea, appearance of edema, etc). At the end of the study, eyes were excised and the physiopathology state was determined for corneal and conjunctival specimens [187]. As a conclusion, it could be stated that no clinical or pathological differences were observed between the eyes treated with CS nanoparticles and the control eyes. With respect to the ocular tolerance of CS nanoparticles complexed with lipids (LCS-nanoparticles), the results were similar to those observed for plain CS nanoparticles [178]. More specifically, the clinical macroscopic sign score was compatible with a non-irritated ocular surface, no histological alterations were observed, and abnormal inflammatory cells were absent in cornea, conjunctiva, and lids, thus indicating no irritation or alterations in the ocular surface structures of the treated rabbits. In the case of HA/CS nanoparticles, their tolerance after acute administration was explored in an identical fashion as described above. The micro and macroscopic evaluation did not show any signs of damage, irritability or dysfunction. Based upon the absence of histological and functional alterations of the ocular surface, the HA/CS nanoparticles appear to be non-hazardous and have excellent in vivo tolerance [191]. In addition, some early studies were performed in order to have preliminary insights into the ocular fate of HA/CS 114 M. de la Fuente et al. / Advanced Drug Delivery Reviews 62 (2010) 100–117 nanoparticles. Essentially, the kinetics of the fluorescent signal associated to HA-fl/CS (hyaluronic acid was covalently labelled with fluoresceinamine) were analysed under the confocal microscope for up to 12 h following their topical administration to rabbits. Images evidenced a decay in the fluorescence intensity over the time. This decay suggested that, somehow, the nanoparticles were bioassimilated into the ocular tissues. This explanation could be supported by the expected degradation of HA and CS. The turnover of HA in the eye could be attributed to the presence of hyaluronidases in the ocular structures [200,201]. Actually, HA-fl in solution, used as a control, rendered to an undetectable fluorescent signal at 4 h after instillation. Similarly, CS was demonstrated to be rapidly degraded by several enzymes [84,202,203], and new degradation routes are being discovered nowadays [86]. These results, together with those referring to the low cytotoxicity in vitro and in vivo, suggest the acceptability of HA/CS nanoparticles by the ocular mucosa [163]. Overall, the results of the cytotoxicity and tolerance of CS-based nanostructures point towards an acceptable toxicological profile of these nanocarriers for topical ocular administration. It may also be presumed that their acceptability, in terms of the dose tolerated, could be dependent on the specific composition of the nanocarriers. In this sense, nanoparticles consisting of CS and HA emerge as a very promising candidate, given their improved interaction with the ocular mucosa and the good safety record of HA in ophthalmology. 7. Conclusions After more than one-decade of track-record reports on the potential of CS for ocular drug delivery leads to some specific conclusions: (i) CS-based nanostructures have a more important ocular retention than CS solutions; (ii) different CS-based nanostructures can be tailored in order to accommodate different types of drugs, from lipophilic low molecular weight compounds (i. e. indomethacin) to medium size peptides (Cyclosporin A) and large gene molecules; (iii) different CS-based nanostructures have shown a capacity to enter the corneal and conjunctival epithelia; (iv) these nanostructures have been found efficacious for increasing the penetration of drugs, i.e. indomethacin, and promoting the transfection of genes in the ocular surface; (v) preliminary data have given indications of the good tolerance and acceptability of these nanocarriers from a toxicological perspective. References [1] C. Bucolo, A. Maltese, F. Drago, When nanotechnology meets the ocular surface, Expert Rev. Ophthalmol. 3 (2008) 325–332. [2] H.B. Raju, J.L. Goldberg, Nanotechnology for ocular therapeutics and tissue repair, Expert Rev. Ophthalmol. 3 (2008) 431–436. [3] A.A. Badawi, H.M. El-Laithy, R.K. El Qidra, H. El Mofty, M. El Dally, Chitosan based nanocarriers for indomethacin ocular delivery, Arch. Pharm. Res. 31 (2008) 1040–1049. [4] J.L. Bourges, S.E. Gautier, F. Delie, R.A. Bejjani, J.C. Jeanny, R. Gurny, D. BenEzra, F.F. Behar-Cohen, Ocular drug delivery targeting the retina and retinal pigment epithelium using polylactide nanoparticles, Invest. Ophthalmol. Vis. Sci. 44 (2003) 3562–3569. [5] A.M. de Campos, Y. Diebold, E.L. Carvalho, A. Sanchez, M.J. Alonso, Chitosan nanoparticles as new ocular drug delivery systems: in vitro stability, in vivo fate, and cellular toxicity, Pharm. Res. 21 (2004) 803–810. [6] A.M. De Campos, A. Sanchez, M.J. Alonso, Chitosan nanoparticles: a new vehicle for the improvement of the delivery of drugs to the ocular surface. Application to cyclosporin A, Int. J. Pharm. 224 (2001) 159–168. [7] M. de la Fuente, N. Csaba, M. Garcia-Fuentes, M.J. Alonso, Nanoparticles as protein and gene carriers to mucosal surfaces, Nanomed 3 (2008) 845–857. [8] O. Kayser, A. Lemke, N. Hernandez-Trejo, The impact of nanobiotechnology on the development of new drug delivery systems, Curr. Pharm. Biotechnol. 6 (2005) 3–5. [9] A. Sanchez, M.J. Alonso, Nanoparticular carriers for ocular drug delivery, in: V.P. Torchilin (Ed.), Nanoparticulates as Drug Carriers, London, 2006, pp. 649–673. [10] R.M. Mainardes, M.C. Urban, P.O. Cinto, N.M. Khalil, M.V. Chaud, R.C. Evangelista, M.P. Gremiao, Colloidal carriers for ophthalmic drug delivery, Curr. Drug Targets 6 (2005) 363–371. [11] J. Vandervoort, A. Ludwig, Ocular drug delivery: nanomedicine applications, Nanomed 2 (2007) 11–21. [12] C. Bloquel, J.L. Bourges, E. Touchard, M. Berdugo, D. BenEzra, F. Behar-Cohen, Non-viral ocular gene therapy: potential ocular therapeutic avenues, Adv. Drug Deliv. Rev. 58 (2006) 1224–1242. [13] T. Borras, Recent developments in ocular gene therapy, Exp. Eye Res. 76 (2003) 643–652. [14] M. Ali, M.E. Byrne, Challenges and solutions in topical ocular drug-delivery systems, Expert Rev. Clin. Pharmacol. 1 (2008) 145–161. [15] C.L. Bourlais, L. Acar, H. Zia, P.A. Sado, T. Needham, R. Leverge, Ophthalmic drug delivery systems—recent advances, Prog. Retin. Eye Res. 17 (1998) 33–58. [16] Y.B. Choy, J.-H. Park, B.E. McCarey, H.F. Edelhauser, M.R. Prausnitz, Mucoadhesive microparticles engineered for ophthalmic drug delivery, J. Phys. Chem. Solids 69 (2008) 1533–1536. [17] R. Gaudana, J. Jwala, S.H.S. Boddu, A.K. Mitra, Recent perspectives in ocular drug delivery, Pharm. Res. 26 (2009) 1197–1216. [18] P.M. Hughes, O. Olejnik, J.E. Chang-Lin, C.G. Wilson, Topical and systemic drug delivery to the posterior segments, Adv. Drug Deliv. Rev. 57 (2005) 2010–2032. [19] D. Meisner, M. Mezei, Liposome ocular delivery systems, Adv. Drug Deliv. Rev. 16 (1995) 75–93. [20] T.F. Vandamme, Microemulsions as ocular drug delivery systems: recent developments and future challenges, Prog. Retin. Eye Res. 21 (2002) 15–34. [21] A. Zimmer, J. Kreuter, Microspheres and nanoparticles used in ocular delivery systems, Adv. Drug Deliv. Rev. 16 (1995) 61–73. [22] C. Losa, M.J. Alonso, J.L. Vila, F. Orallo, J. Martinez, J.A. Saavedra, J.C. Pastor, Reduction of cardiovascular side effects associated with ocular administration of metipranolol by inclusion in polymeric nanocapsules, J. Ocul. Pharmacol. 8 (1992) 191–198. [23] C. Losa, P. Calvo, E. Castro, J.L. Vila-Jato, M.J. Alonso, Improvement of ocular penetration of amikacin sulphate by association to poly(butylcyanoacrylate) nanoparticles, J. Pharm. Pharmacol. 43 (1991) 548–552. [24] C. Losa, L. Marchal-Heussler, F. Orallo, J.L. Vila Jato, M.J. Alonso, Design of new formulations for topical ocular administration: polymeric nanocapsules containing metipranolol, Pharm. Res. 10 (1993) 80–87. [25] P. Calvo, M.J. Alonso, J.L. Vila-Jato, J.R. Robinson, Improved ocular bioavailability of indomethacin by novel ocular drug carriers, J. Pharm. Pharmacol. 48 (1996) 1147–1152. [26] P. Calvo, A. Sanchez, J. Martinez, M.I. Lopez, M. Calonge, J.C. Pastor, M.J. Alonso, Polyester nanocapsules as new topical ocular delivery systems for cyclosporin A, Pharm. Res. 13 (1996) 311–315. [27] P. Calvo, J.L. Vila-Jato, M.J. Alonso, Comparative in vitro evaluation of several colloidal systems, nanoparticles, nanocapsules, and nanoemulsions, as ocular drug carriers, J. Pharm. Sci. 85 (1996) 530–536. [28] P. Calvo, J.L. Vila-Jato, M.J. Alonso, Evaluation of cationic polymer-coated nanocapsules as ocular drug carriers, Int. J. Pharm. 153 (1997) 41–50. [29] A.M. De Campos, A. Sanchez, R. Gref, P. Calvo, M.J. Alonso, The effect of a PEG versus a chitosan coating on the interaction of drug colloidal carriers with the ocular mucosa, Eur. J. Pharm. Sci. 20 (2003) 73–81. [30] M.J. Alonso, A. Sanchez, The potential of chitosan in ocular drug delivery, J. Pharm. Pharmacol. 55 (2003) 1451–1463. [31] O. Felt, P. Furrer, J.M. Mayer, B. Plazonnet, P. Buri, R. Gurny, Topical use of chitosan in ophthalmology: tolerance assessment and evaluation of precorneal retention, Int. J. Pharm. 180 (1999) 185–193. [32] C.M. Lehr, J.A. Bouwstra, E.H. Schacht, H.E. Junginger, In vitro evaluation of mucoadhesive properties of chitosan and some other natural polymers, Int. J. Pharm. 78 (1992) 43–48. [33] N.G.M. Schipper, S. Olsson, J.A. Hoogstraate, A.G. DeBoer, K.M. Varum, P. Artursson, Chitosans as absorption enhancers for poorly absorbable drugs 2: mechanism of absorption enhancement, Pharm. Res. 14 (1997) 923–929. [34] P. Calvo, C. Remuñan-Lopez, J.L. Vila-Jato, M.J. Alonso, Chitosan and chitosan/ ethylene oxide-propylene oxide block copolymer nanoparticles as novel carriers for proteins and vaccines, Pharm. Res. 14 (1997) 1431–1436. [35] M. de la Fuente, B. Seijo, M.J. Alonso, Novel hyaluronan-based nanocarriers for transmucosal delivery of macromolecules, Macromol. Biosci. 8 (2008) 441–450. [36] F. Maestrelli, M. Garcia-Fuentes, P. Mura, M.J. Alonso, A new drug nanocarrier consisting of chitosan and hydoxypropylcyclodextrin, Eur. J. Pharm. Biopharm. 63 (2006) 79–86. [37] N. Csaba, M. Koping-Hoggard, E. Fernandez-Megia, R. Novoa-Carballal, R. Riguera, M.J. Alonso, Ionically crosslinked chitosan nanoparticles as gene delivery systems: effect of PEGylation degree on in vitro and in vivo gene transfer, J. Biomed. Nanotech. 5 (2009) 162–171. [38] M. De La Fuente, B. Seijo, M.J. Alonso, Novel hyaluronic acid-chitosan nanoparticles for ocular gene therapy, Invest. Ophthalmol. Vis. Sci. 49 (2008) 2016–2024. [39] K.K. Gukasyan HJ, Lee VHL, The conjunctival barrier in ocular drug delivery in: Springer U.S. (Eds.), Drug Absorption Studies, vol.7, 2008, pp.307–320. [40] K. Hosoya, V.H. Lee, K.J. Kim, Roles of the conjunctiva in ocular drug delivery: a review of conjunctival transport mechanisms and their regulation, Eur. J. Pharm. Biopharm. 60 (2005) 227–240. [41] J. Barar, A.R. Javadzadeh, Y. Omidi, Ocular novel drug delivery: impacts of membranes and barriers, Expert Opin. Drug Deliv. 5 (2008) 567–581. [42] C. Le Bourlais, L. Acar, H. Zia, P.A. Sado, T. Needham, R. Leverge, Ophthalmic drug delivery systems — recent advances, Progr. Retin. Eye Res. 17 (1998) 33–58. [43] S. Duvvuri, S. Majumdar, A.K. Mitra, Role of metabolism in ocular drug delivery, Curr. Drug Metab. 5 (2004) 507–515. [44] A. Urtti, Challenges and obstacles of ocular pharmacokinetics and drug delivery, Adv. Drug Deliv. Rev. 58 (2006) 1131–1135. M. de la Fuente et al. / Advanced Drug Delivery Reviews 62 (2010) 100–117 [45] E. Mannermaa, K.S. Vellonen, A. Urtti, Drug transport in corneal epithelium and blood-retina barrier: emerging role of transporters in ocular pharmacokinetics, Adv. Drug Deliv. Rev. 58 (2006) 1136–1163. [46] H.S. Gunda S, Mandava N and Mitra AK, Barriers in ocular drug delivery, in: Human Press (Eds.), Ocular Transporters in Opthalmic Diseases and Drug Delivery, 2008, pp.399–413. [47] P. Paolicelli, M. de la Fuente, A. Sanchez, B. Seijo, M.J. Alonso, Chitosan nanoparticles for drug delivery to the eye, Expert Opin. Drug Deliv. 6 (2009) 239–253. [48] J.C. Lang, Ocular drug delivery conventional ocular formulations, Adv. Drug Deliv. Rev. 16 (1995) 39–43. [49] I. Ahmed, T.F. Patton, Disposition of timolol and inulin in the rabbit eye following corneal versus non-corneal absorption, Int. J. Pharm. 38 (1987) 9–21. [50] K. Jarvinen, T. Jarvinen, A. Urtti, Ocular absorption following topical delivery, Adv. Drug Deliv. Rev. 16 (1995) 3–19. [51] M.R. Prausnitz, A. Edwards, J.S. Noonan, D.E. Rudnick, H.F. Edelhauser, D.H. Geroski, Measurement and prediction of transient transport across sclera for drug delivery to the eye, Ind. Eng. Chem. Res. 37 (1998) 2903–2907. [52] D. Ghate, H.F. Edelhauser, Ocular drug delivery, Expert Opin. Drug Deliv. 3 (2006) 275–287. [53] K.M. Hamalainen, K. Kananen, S. Auriola, K. Kontturi, A. Urtti, Characterization of paracellular and aqueous penetration routes in cornea, conjunctiva, and sclera, Invest. Ophthalmol. Vis. Sci. 38 (1997) 627–634. [54] B. Gumbiner, Structure, biochemistry, and assembly of epithelial tight junctions, Am. J. Physiol. Cell Physiol. 253 (1987). [55] A.J.W. Huang, S.C.G. Tseng, K.R. Kenyon, Paracellular permeability of corneal and conjunctival epithelia, Invest. Ophthalmol. Vis. Sci. 30 (1989) 684–689. [56] V.H.L. Lee, Precorneal, corneal and postcorneal factors, in: A.K. Mitra (Ed.), Ophthalmic Drug Delivery Systems, 1993, pp. 59–81, New York: Marcel Dekker Inc. [57] T.N. Yusifov, A.R. Abduragimov, K. Narsinh, O.K. Gasymov, B.J. Glasgow, Tear lipocalin is the major endonuclease in tears, Mol. Vis. 14 (2008) 180–188. [58] M. Berdugo, F. Valamanesh, C. Andrieu, C. Klein, D. Benezra, Y. Courtois, F. BeharCohen, Delivery of antisense oligonucleotide to the cornea by iontophoresis, Antisense Nucleic Acid Drug Dev. 13 (2003) 107–114. [59] M.F. Cordeiro, A. Mead, R.R. Ali, R.A. Alexander, S. Murray, C. Chen, C. YorkDefalco, N.M. Dean, G.S. Schultz, P.T. Khaw, Novel antisense oligonucleotides targeting TGB-β inhibit in vivo scarring and improve surgical outcome, Gene Ther. 10 (2003) 59–71. [60] S. Duvvuri, S. Majumdar, A.K. Mitra, Drug delivery to the retina: challenges and opportunities, Expert Opin. Biol. Ther. 3 (2003) 45–56. [61] R. Naik, A. Mukhopadhyay, M. Ganguli, Gene delivery to the retina: focus on nonviral approaches, Drug Discov. Today 14 (2009) 306–315. [62] L. Peeters, N.N. Sanders, K. Braeckmans, K. Boussery, J. Van De Voorde, S.C. De Smedt, J. Demeester, Vitreous: a barrier to nonviral ocular gene therapy, Invest. Ophthalmol. Vis. Sci. 46 (2005) 3553–3561. [63] L. Pitkanen, M. Ruponen, J. Nieminen, A. Urtti, Vitreous is a barrier in nonviral gene transfer by cationic lipids and polymers, Pharm. Res. 20 (2003) 576–583. [64] M.B. Bally, P. Harvie, F.M.P. Wong, S. Kong, E.K. Wasan, D.L. Reimer, Biological barriers to cellular delivery of lipid-based DNA carriers, Adv. Drug Deliv. Rev. 38 (1999) 291–315. [65] M.D. Hughes, M. Hussain, Q. Nawaz, P. Sayyed, S. Akhtar, The cellular delivery of antisense oligonucleotides and ribozymes, Drug Discov. Today 6 (2001) 303–315. [66] F. Liu, L. Huang, Development of non-viral vectors for systemic gene delivery, J. Control. Release 78 (2002) 259–266. [67] C.W. Pouton, L.W. Seymour, Key issues in non-viral gene delivery, Adv. Drug Deliv. Rev. 46 (2000) 187–203. [68] H. Kamiya, H. Tsuchiya, J. Yamazaki, H. Harashima, Intracellular trafficking and transgene expression of viral and non-viral gene vectors, Adv. Drug Deliv. Rev. 52 (2001) 153–164. [69] T.F. Patton, J.R. Robinson, Ocular evaluation of polyvinyl alcohol vehicle in rabbits, J. Pharm. Sci. 64 (1975) 1312–1316. [70] I.P. Kaur, R. Smitha, Penetration enhancers and ocular bioadhesives: two new avenues for ophthalmic drug delivery, Drug Dev. Ind. Pharm. 28 (2002) 353–369. [71] A. Ludwig, The use of mucoadhesive polymers in ocular drug delivery, Adv. Drug Deliv. Rev. 57 (2005) 1595–1639. [72] I. Henriksen, K.L. Green, J.D. Smart, G. Smistad, J. Karlsen, Bioadhesion of hydrated chitosans: an in vitro and in vivo study, Int. J. Pharm. 145 (1996) 231–240. [73] E.E. Hassan, J.M. Gallo, A simple rheological method for the in vitro assessment of mucin-polymer bioadhesive bond strength, Pharm. Res. 7 (1990) 491–495. [74] I. Sogias, A. Williams, V. Khutoryanskiy, Why is chitosan mucoadhesive? Biomacromolecules 9 (2008) 1837–1842. [75] P. Artursson, T. Lindmark, S.S. Davis, L. Illum, Effect of chitosan on the permeability of monolayers of intestinal epithelial cells (Caco-2), Pharm. Res. 11 (1994) 1358–1361. [76] S.M. Van Der Merwe, J.C. Verhoef, J.H.M. Verheijden, A.F. Kotze, H.E. Junginger, Trimethylated chitosan as polymeric absorption enhancer for improved peroral delivery of peptide drugs, Eur. J. Pharm. Biopharm. 58 (2004) 225–235. [77] V. Dodane, M. Amin Khan, J.R. Merwin, Effect of chitosan on epithelial permeability and structure, Int. J. Pharm. 182 (1999) 21–32. [78] M. Mucha, Rheological characteristics of semi-dilute chitosan solutions, Macromol. Chem. Phys. 198 (1997) 471–484. [79] W. Wang, D. Xu, Viscosity and flow properties of concentrated solutions of chitosan with different degrees of deacetylation, Int. J. Biol. Macromol. 16 (1994) 149–152. 115 [80] J.M. Tiffany, The viscosity of human tears, Int. Ophthalmol. 15 (1991) 371–376. [81] J.L. Greaves, O. Olejnik, C.G. Wilson, Polymers and the precorneal tear film, S.T.P. Pharma Sci. 2 (1992) 13–33. [82] G. Di Colo, Y. Zambito, S. Burgalassi, I. Nardini, M.F. Saettone, Effect of chitosan and of n-carboxymethylchitosan on intraocular penetration of topically applied ofloxacin, Int. J. Pharm. 273 (2004) 37–44. [83] K.M. Varum, M.M. Myhr, R.J.N. Hjerde, O. Smidsrod, In vitro degradation rates of partially n-acetylated chitosans in human serum, Carbohydr. Res. 299 (1997) 99–101. [84] R.A. Muzzarelli, Human enzymatic activities related to the therapeutic administration of chitin derivatives, Cell. Mol. Life Sci. 53 (1997) 131–140. [85] A. Temel, H. Kazokoglu, Y. Taga, Tear lysozyme levels in contact lens wearers, Ann. Ophthalmol. 23 (1991) 191–194. [86] C. Gorzelanny, B. Poeppelmann, S. Haebel, B.M. Moerschbacher, S.W. Schneider, Human chitotriosidase degradation of chitosan and chitin and its role within the innate immune response, Proceedings of the 9th International Conference of The European Chitin Society (2009) pp. 116–117. [87] E.I. Rabea, M.E.T. Badawy, C.V. Stevens, G. Smagghe, W. Steurbaut, Chitosan as antimicrobial agent: applications and mode of action, Biomacromolecules 4 (2003) 1457–1465. [88] O. Felt, A. Carrel, P. Baehni, P. Buri, R. Gurny, Chitosan as tear substitute: a wetting agent endowed with antimicrobial efficacy, J. Ocular Pharmacol. Ther. 16 (2000) 261–270. [89] O. Felt, V. Baeyens, P. Buri, R. Gurny, Delivery of antibiotics to the eye using a positively charged polysaccharide as vehicle, A.A.P.S. Pharm. Sci. 3 (2001) 1–7. [90] Y. Cao, C. Zhang, W. Shen, Z. Cheng, L. Yu, Q. Ping, Poly(N-isopropylacrylamide)chitosan as thermosensitive in situ gel-forming system for ocular drug delivery, J. Control. Release 120 (2007) 186–194. [91] H. Gupta, S. Jain, R. Mathur, P. Mishra, A.K. Mishra, T. Velpandian, Sustained ocular drug delivery from a temperature and pH triggered novel in situ gel system, Drug Deliv. 14 (2007) 507–515. [92] E.M. del Amo, A. Urtti, Current and future ophthalmic drug delivery systems. A shift to the posterior segment, Drug Discov. Today 13 (2008) 135–143. [93] E. Fattal, A. Bochot, Ocular delivery of nucleic acids: antisense oligonucleotides, aptamers and siRNA, Adv. Drug Deliv. Rev. 58 (2006) 1203–1223. [94] S.K. Sahoo, F. Dilnawaz, S. Krishnakumar, Nanotechnology in ocular drug delivery, Drug Discov. Today 13 (2008) 144–151. [95] H. Yang, R. Wang, Q. Gu, X. Zhang, Feasibility study of chitosan as intravitreous tamponade material, Graefes Arch. Clin. Exper. Ophthalmol. 246 (2008) 1097–1105. [96] T.W. Prow, I. Bhutto, S.Y. Kim, R. Grebe, C. Merges, D.S. McLeod, K. Uno, M. Mennon, L. Rodriguez, K. Leong, G.A. Lutty, Ocular nanoparticle toxicity and transfection of the retina and retinal pigment epithelium, Nanomedicine 4 (2008) 340–349. [97] J. Alsenz, M. Kansy, High throughput solubility measurement in drug discovery and development, Adv. Drug Deliv. Rev. 59 (2007) 546–567. [98] S. Tamilvanan, S. Benita, The potential of lipid emulsion for ocular delivery of lipophilic drugs, Eur. J. Pharm. Biopharm. 58 (2004) 357–368. [99] E. Elbaz, Positively charged submicron emulsions — a new type of colloidal drug carrier, Int. J. Pharm. 96 (1993). [100] S.H. Klang, J. Frucht-Pery, A. Hoffman, S. Benita, Physicochemical characterization and acute toxicity evaluation of a positively-charged submicron emulsion vehicle, J. Pharm. Pharmacol. 46 (1994) 986–993. [101] S. Klang, M. Abdulrazik, S. Benita, Influence of emulsion droplet surface charge on indomethacin ocular tissue distribution, Pharm. Dev. Technol. 5 (2000) 521–532. [102] S. Tamilvanan, K. Khoury, D. Gilhar, S. Benita, Ocular delivery of cyclosporin A I. Design and characterization of cyclosporin a-loaded positively-charged submicron emulsion, S.T.P. Pharma Sci. 11 (2001) 421–426. [103] M. Abdulrazik, S. Tamilvanan, K. Khoury, S. Benita, Ocular delivery of cyclosporin A II. Effect of submicron emulsion's surface charge on ocular distribution of topical cyclosporin a, S.T.P. Pharma Sci. 11 (2001) 427–432. [104] L. Payet, E.M. Terentjev, Emulsification and stabilization mechanisms of o/w emulsions in the presence of chitosan, Langmuir 24 (2008) 12247–12252. [105] M. Jumaa, B.W. Muller, Physicochemical properties of chitosan-lipid emulsions and their stability during the autoclaving process, Int. J. Pharm. 183 (1999) 175–184. [106] P. Calvo, C. Remuñan-Lopez, J.L. Vila-Jato, M.J. Alonso, Development of positively charged colloidal drug carriers: chitosan-coated polyester nanocapsules and submicron-emulsions, Colloid Polym. Sci. 275 (1997) 46–53. [107] L. Marchal-Heussler, H. Fessi, J.P. Devissaguet, M. Hoffman, T.P. Maincen, Colloidal drug delivery systems for the eye. A comparison of the efficacy of three different polymers: polyisobutylcyanoacrylate, polylactic-co-glycolic acid, and poly-epsilon-caprolacton, S.T.P. Pharma Sci. 2 (1992) 98–104. [108] D. Aggarwal, I.P. Kaur, Improved pharmacodynamics of timolol maleate from a mucoadhesive niosomal ophthalmic drug delivery system, Int. J. Pharm. 290 (2005) 155–159. [109] X.B. Yuan, Y.B. Yuan, W. Jiang, J. Liu, E.J. Tian, H.M. Shun, D.H. Huang, X.Y. Yuan, H. Li, J. Sheng, Preparation of rapamycin-loaded chitosan/PLA nanoparticles for immunosuppression in corneal transplantation, Int. J. Pharm. 349 (2008) 241–248. [110] I.F. Uchegbu, L. Sadiq, M. Arastoo, A.I. Gray, W. Wang, R.D. Waigh, A.G. Schatzlein, Quaternary ammonium palmitoyl glycol chitosan—a new polysoap for drug delivery, Int. J. Pharm. 224 (2001) 185–199. [111] X. Qu, V.V. Khutoryanskiy, A. Stewart, S. Rahman, B. Papahadjopoulos-Sternberg, C. Dufes, D. McCarthy, C.G. Wilson, R. Lyons, K.C. Carter, A. Schatzlein, I.F. Uchegbu, 116 [112] [113] [114] [115] [116] [117] [118] [119] [120] [121] [122] [123] [124] [125] [126] [127] [128] [129] [130] [131] [132] [133] [134] [135] [136] [137] [138] [139] [140] [141] M. de la Fuente et al. / Advanced Drug Delivery Reviews 62 (2010) 100–117 Carbohydrate-based micelle clusters which enhance hydrophobic drug bioavailability by up to 1 order of magnitude, Biomacromolecules 7 (2006) 3452–3459. X.B. Yuan, H. Li, Y.B. Yuan, Preparation of cholesterol-modified chitosan selfaggregated nanoparticles for delivery of drugs to ocular surface, Carbohydr. Polym. 65 (2006) 337–345. F. Sonvico, A. Cagnani, A. Rossi, S. Motta, M.T. Di Bari, F. Cavatorta, M.J. Alonso, A. Deriu, P. Colombo, Formation of self-organized nanoparticles by lecithin/ chitosan ionic interaction, Int. J. Pharm. 324 (2006) 67–73. E.M.M. Del Valle, Cyclodextrins and their uses: a review, Proc. Biochem. 39 (2004) 1033–1046. V.J. Stella, R.A. Rajewski, Cyclodextrins: their future in drug formulation and delivery, Pharm. Res. 14 (1997) 556–567. D. Teijeiro-Osorio, C. Remunan-Lopez, M.J. Alonso, New generation of hybrid poly/oligosaccharide nanoparticles as carriers for the nasal delivery of macromolecules, Biomacromolecules 10 (2008) 243–249. D. Teijeiro-Osorio, C. Remunan-Lopez, M.J. Alonso, Chitosan/cyclodextrin nanoparticles can efficiently transfect the airway epithelium in vitro, Eur. J. Pharm. Biopharm. 71 (2008) 257–263. T. Loftssona, T. Jarvinen, Cyclodextrins in ophthalmic drug delivery, Adv. Drug Deliv. Rev. 36 (1999) 59–79. W.W. Hauswirth, L. Beaufrere, Ocular gene therapy: Quo vadis? Invest. Ophthalmol. Vis. Sci. 41 (2000) 2821–2826. U. Pleyer, T. Ritter, Gene therapy in immune-mediated diseases of the eye, Prog. Retin. Eye Res. 22 (2003) 277–293. S.J. Reich, J. Bennett, Gene therapy for ocular neovascularization: a cure in sight, Curr. Opin. Genet. Dev. 13 (2003) 317–322. E. Toropainen, M. Hornof, K. Kaarniranta, P. Johansson, A. Urtti, Corneal epithelium as a platform for secretion of transgene products after transfection with liposomal gene eyedrops, J. Gene Med. 9 (2007) 208–216. M. Hornof, M. de la Fuente, M. Hallikainen, R.H. Tammi, A. Urtti, Low molecular weight hyaluronan shielding of DNA/PEI polyplexes facilitates CD44 receptor mediated uptake in human corneal epithelial cells, J. Gene Med. 10 (2008) 70–80. T. Hudde, S.A. Rayner, R.M. Comer, M. Weber, J.D. Isaacs, H. Waldmann, D.F. Larkin, A.J. George, Activated polyamidoamine dendrimers, a non-viral vector for gene transfer to the corneal endothelium, Gene Ther. 6 (1999) 939–943. U. Pleyer, D. Groth, B. Hinz, O. Keil, E. Bertelmann, P. Rieck, R. Reszka, Efficiency and toxicity of liposome-mediated gene transfer to corneal endothelial cells, Exp. Eye Res. 73 (2001) 1–7. G. Borchard, Chitosans for gene delivery, Adv. Drug Deliv. Rev. 52 (2001) 145–150. H.H. Chen, Y.P. Ho, X. Jiang, H.Q. Mao, T.H. Wang, K.W. Leong, Quantitative comparison of intracellular unpacking kinetics of polyplexes by a model constructed from quantum dot-fret, Mol. Ther. 16 (2008) 324–332. F.C. MacLaughlin, R.J. Mumper, J. Wang, J.M. Tagliaferri, I. Gill, M. Hinchcliffe, A.P. Rolland, Chitosan and depolymerized chitosan oligomers as condensing carriers for in vivo plasmid delivery, J. Control. Release 56 (1998) 259–272. H.Q. Mao, K. Roy, V.L. Troung-Le, K.A. Janes, K.Y. Lin, Y. Wang, J.T. August, K.W. Leong, Chitosan–DNA nanoparticles as gene carriers: synthesis, characterization and transfection efficiency, J. Control. Release 70 (2001) 399–421. T. Sato, T. Ishii, Y. Okahata, In vitro gene delivery mediated by chitosan. Effect of pH, serum, and molecular mass of chitosan on the transfection efficiency, Biomaterials 22 (2001) 2075–2080. T. Ishii, Y. Okahata, T. Sato, Mechanism of cell transfection with plasmid/chitosan complexes, Biochim. Biophys. Acta 1514 (2001) 51–64. P. Erbacher, S. Zou, T. Bettinger, A.M. Steffan, J.S. Remy, Chitosan-based vector/ DNA complexes for gene delivery: biophysical characteristics and transfection ability, Pharm. Res. 15 (1998) 1332–1339. K. Turan, K. Nagata, Chitosan–DNA nanoparticles: the effect of cell type and hydrolysis of chitosan on in vitro DNA transfection, Pharm. Dev. Technol. 11 (2006) 503–512. X. Liu, K.A. Howard, M. Dong, M.O. Andersen, U.L. Rahbek, M.G. Johnsen, O.C. Hansen, F. Besenbacher, J. Kjems, The influence of polymeric properties on chitosan/siRNA nanoparticle formulation and gene silencing, Biomaterials 28 (2007) 1280–1288. S.C. Richardson, H.V. Kolbe, R. Duncan, Potential of low molecular mass chitosan as a DNA delivery system: biocompatibility, body distribution and ability to complex and protect DNA, Int. J. Pharm. 178 (1999) 231–243. M. Koping-Hoggard, K.M. Varum, M. Issa, D. Danielsen, B.E. Christenses, B.T. Stokke, P. Artursson, Improved chitosan-mediated gene delivery based on easily dissociated chitosan polyplexes of highly defined chitosan oligomers, Gene Ther. 11 (2004) 1441–1452. K. Regnstrom, E.G. Ragnarsson, M. Fryknas, M. Koping-Hoggard, P. Artursson, Gene expression profiles in mouse lung tissue after administration of two cationic polymers used for nonviral gene delivery, Pharm. Res. 23 (2006) 475–482. M. Koping-Hoggard, I. Tubulekas, H. Guan, K. Edwards, M. Nilsson, K.M. Varum, P. Artursson, Chitosan as a nonviral gene delivery system. Structure–property relationships and characteristics compared with polyethylenimine in vitro and after lung administration in vivo, Gene Ther. 8 (2001) 1108–1121. K.A. Howard, U.L. Rahbek, X. Liu, C.K. Damgaard, S.Z. Glud, M.O. Andersen, M.B. Hovgaard, A. Schmitz, J.R. Nyengaard, F. Besenbacher, J. Kjems, RNA interference in vitro and in vivo using a chitosan/siRNA nanoparticle system, Mol. Ther. 14 (2006) 476–484. M. Ruponen, P. Honkakoski, S. Ronkko, J. Pelkonen, M. Tammi, A. Urtti, Extracellular and intracellular barriers in non-viral gene delivery, J. Control. Release 93 (2003) 213–217. M. Ruponen, P. Honkakoski, M. Tammi, A. Urtti, Cell-surface glycosaminoglycans inhibit cation-mediated gene transfer, J Gene Med. 6 (2004) 405–414. [142] S. Danielsen, S. Strand, C. de Lange Davies, B.T. Stokke, Glycosaminoglycan destabilization of DNA–chitosan polyplexes for gene delivery depends on chitosan chain length and GAG properties, Biochim. Biophys. Acta 1721 (2005) 44–54. [143] N. Csaba, M. Koping-Hoggard, M.J. Alonso, Ionically crosslinked chitosan/ tripolyphosphate nanoparticles for oligonucleotide and plasmid DNA delivery International Journal of Pharmaceutics, Int. J. Pharm. 382 (2009) 205–214. [144] K.A. Janes, P. Calvo, M.J. Alonso, Polysaccharide colloidal particles as delivery systems for macromolecules, Adv. Drug Deliv. Rev. 47 (2001) 83–97. [145] Q. Gan, T. Wang, C. Cochrane, P. McCarron, Modulation of surface charge, particle size and morphological properties of chitosan–TPP nanoparticles intended for gene delivery, Colloids Surf. B Biointerfaces 44 (2005) 65–73. [146] T.H. Dung, S.R. Lee, S.D. Han, S.J. Kim, Y.M. Ju, M.S. Kim, H. Yoo, Chitosan–TPP nanoparticle as a release system of antisense oligonucleotide in the oral environment, J. Nanosci. Nanotechnol. 7 (2007) 3695–3699. [147] H. Katas, H.O. Alpar, Development and characterisation of chitosan nanoparticles for siRNA delivery, J. Control. Release 115 (2006) 216–225. [148] S.L. Wang, H.H. Yao, L.L. Guo, L. Dong, S.G. Li, Y.P. Gu, Z.H. Qin, Selection of optimal sites for TGFB1 gene silencing by chitosan–TPP nanoparticle-mediated delivery of shRNA, Cancer Genet. Cytogenet. 190 (2009) 8–14. [149] J. Chen, W.L. Yang, G. Li, J. Qian, J.L. Xue, S.K. Fu, D.R. Lu, Transfection of mepo gene to intestinal epithelium in vivo mediated by oral delivery of chitosan–DNA nanoparticles, World J. Gastroenterol. 10 (2004) 112–116. [150] K. Khatri, A.K. Goyal, P.N. Gupta, N. Mishra, S.P. Vyas, Plasmid DNA loaded chitosan nanoparticles for nasal mucosal immunization against hepatitis B, Int. J. Pharm. 354 (2008) 235–241. [151] M. Bivas-Benita, M. Laloup, S. Versteyhe, J. Dewit, J. De Braekeleer, E. Jongert, G. Borchard, Generation of toxoplasma gondii GRA1 protein and DNA vaccine loaded chitosan particles: preparation, characterization, and preliminary in vivo studies, Int. J. Pharm. 266 (2003) 17–27. [152] L.J.A.L. Lapcik, L. Lapcik, S. De Smedt, J. Demeester, P. Chabrecek, Hyaluronan: preparation, structure, properties, and applications, Chem. Rev. 98 (1998) 2663–2684. [153] M. Cobo, N. Beaty, Vitrax (sodium hyaluronate) in anterior segment surgery: a review and clinical study summary, Adv. Ther. 7 (1990) 51–60. [154] G.D. Prestwich, J.W. Kuo, Chemically-modified HA for therapy and regenerative medicine, Curr. Pharm. Biotechnol. 9 (2008) 242–245. [155] J.L. Greaves, C.G. Wilson, Treatment of diseases of the eye with mucoadhesive delivery systems, Adv. Drug Deliv. Rev. 11 (1993) 349–383. [156] R.E. Eliaz, F.C. Szoka Jr., Liposome-encapsulated doxorubicin targeted to CD44: a strategy to kill CD44-overexpressing tumor cells, Cancer Res. 61 (2001) 2592–2601. [157] R.E. Eliaz, S. Nir, F.C. Szoka Jr., Interactions of hyaluronan-targeted liposomes with cultured cells: modeling of binding and endocytosis, Methods Enzymol. 387 (2004) 16–33. [158] S. Barbault-Foucher, R. Gref, P. Russo, J. Guechot, A. Bochot, Design of polyepsilon-caprolactone nanospheres coated with bioadhesive hyaluronic acid for ocular delivery, J. Control. Release 83 (2002) 365–375. [159] P. Aragona, Hyaluronan in the treatment of ocular surface disorders, in: H.G. Garga, C.A. Hales (Eds.), Chemistry and Biology of Hyaluronan, Elsevier Ltd, Oxford, UK, 2004, pp. 529–551. [160] P. Fagerholm, Endogenous hyaluronan in the anterior segment of the eye, Progr. Retin. Eye Res. 15 (1996) 281–296. [161] L.E. Lerner, D.M. Schwartz, D.G. Hwang, E.L. Howes, R. Stern, Hyaluronan and CD44 in the human cornea and limbal conjunctiva, Exp. Eye Res. 67 (1998) 481–484. [162] U.B. Kompella, S. Sundaram, S. Raghava, E.R. Escobar, Luteinizing hormonereleasing hormone agonist and transferrin functionalizations enhance nanoparticle delivery in a novel bovine ex vivo eye model, Mol. Vis. 12 (2006) 1185–1198. [163] M. de la Fuente, B. Seijo, M.J. Alonso, Bioadhesive hyaluronan–chitosan nanoparticles can transport genes across the ocular mucosa and transfect ocular tissue, Gene Ther. 15 (2008) 668–676. [164] M. de la Fuente, B. Seijo, M.J. Alonso, Design of novel polysaccharidic nanostructures for gene delivery, Nanotechnology 19 (2008) 1–9. [165] M. Raviña, E. Fernandez-Megia, R. Novoa, R. Riguera, M.J. Alonso, A. Sanchez, Chitosan–PEG/hyaluronic acid nanoparticles as nanocarriers for siRNA, Cellular Delivery of Therapeutic Macromolecules International Symposium, 2008, p. 54. [166] M. Ruponen, S. Ronkko, P. Honkakoski, J. Pelkonen, M. Tammi, A. Urtti, Extracellular glycosaminoglycans modify cellular trafficking of lipoplexes and polyplexes, J. Biol. Chem. 276 (2001) 33875–33880. [167] T. Ito, N. Iida-Tanaka, T. Niidome, T. Kawano, K. Kubo, K. Yoshikawa, T. Sato, Z. Yang, Y. Koyama, Hyaluronic acid and its derivative as a multi-functional gene expression enhancer: protection from non-specific interactions, adhesion to targeted cells, and transcriptional activation, J. Control. Release 112 (2006) 382–388. [168] E. Toropainen, V.P. Ranta, A. Talvitie, P. Suhonen, A. Urtti, Culture model of human corneal epithelium for prediction of ocular drug absorption, Invest. Ophthalmol. Vis. Sci. 42 (2001) 2942–2948. [169] M. Hornof, E. Toropainen, A. Urtti, Cell culture models of the ocular barriers, Eur. J. Pharm. Biopharm. 60 (2005) 207–225. [170] Z. Liu, J. Li, S. Nie, H. Liu, P. Ding, W. Pan, Study of an alginate/HPMC-based in situ gelling ophthalmic delivery system for gatifloxacin, Int. J. Pharm. 315 (2006) 12–17. [171] W. Liu, M. Griffith, F. Li, Alginate microsphere-collagen composite hydrogel for ocular drug delivery and implantation, J. Mater. Sci. Mater. Med. 19 (2008) 3365–3371. M. de la Fuente et al. / Advanced Drug Delivery Reviews 62 (2010) 100–117 [172] R.M. Gilhotra, D.N. Mishra, Alginate-chitosan film for ocular drug delivery: effect of surface cross-linking on film properties and characterization, Pharmazie 63 (2008) 576–579. [173] R.A., B. Sarmento, F. Veiga, D. Ferreira, R. Beufeld, Oral bioavailability of insulina contained in polysaccharidic nanoparticles, Biomacromolecules 8 (2007) 3054–3060. [174] F. Goycoolea, G. Lollo, M.J. Alonso, Chitosan-alginate blended nanoparticles as carriers for transmucosal delivery of macromolecules, Biomacromolecules 10 (2009) 1736–1743. [175] F.M. Goycoolea, I. Higuera-Ciapara, M.J. Alonso, Chitosan–polysaccharide blended nanoparticles for controlled drug delivery, in: R. Reis (Ed.), Handbook on Natural-based Polymers for Biomedical Applications, Cambridge, U.K., 2008, pp. 544–679. [176] S.K. Motwani, S. Chopra, S. Talegaonkar, K. Kohli, F.J. Ahmad, R.K. Khar, Chitosan– sodium alginate nanoparticles as submicroscopic reservoirs for ocular delivery: formulation, optimisation and in vitro characterisation, Eur. J. Pharm. Biopharm. 68 (2008) 513–525. [177] K.L. Douglas, C.A. Piccirillo, M. Tabrizian, Effects of alginate inclusion on the vector properties of chitosan-based nanoparticles, J. Control. Release 115 (2006) 354–361. [178] Y. Diebold, M. Jarrin, V. Saez, E.L. Carvalho, M. Orea, M. Calonge, B. Seijo, M.J. Alonso, Ocular drug delivery by liposome–chitosan nanoparticle complexes (LCS-NP), Biomaterials 28 (2007) 1553–1564. [179] B. Seijo, E.L.S. Carvalho, M.J. Alonso, Chitosan nanoparticles–phospholipid complexes for oral administration of insulin, Proceedings of 4th World Meeting ADRITELF/APGI/APV, 2002, pp. 797–798. [180] Y.Z. Huang, J.Q. Gao, W.Q. Liang, S. Nakagawa, Preparation and characterization of liposomes encapsulating chitosan nanoparticles, Biol. Pharm. Bull. 28 (2005) 387–390. [181] A. Grenha, C. Remunan-Lopez, E.L. Carvalho, B. Seijo, Microspheres containing lipid/chitosan nanoparticles complexes for pulmonary delivery of therapeutic proteins, Eur. J. Pharm. Biopharm. 69 (2008) 83–93. [182] I. Masuda, T. Matsuo, T. Yasuda, N. Matsuo, Gene transfer with liposomes to the intraocular tissues by different routes of administration, Invest. Ophthalmol. Vis. Sci. 37 (1996) 1914–1920. [183] V. Hartmann, S. Keipert, Physico-chemical, in vitro and in vivo characterisation of polymers for ocular use, Pharmazie 55 (2000) 440–443. [184] S. Majumdar, K. Hippalgaonkar, M.A. Repka, Effect of chitosan, benzalkonium chloride and ethylenediaminetetraacetic acid on permeation of acyclovir across isolated rabbit cornea, Int. J. Pharm. 348 (2008) 175–178. [185] A.M. Sadeghi, F.A. Dorkoosh, M.R. Avadi, M. Weinhold, A. Bayat, F. Delie, R. Gurny, B. Larijani, M. Rafiee-Tehrani, H.E. Junginger, Permeation enhancer effect of chitosan and chitosan derivatives: comparison of formulations as soluble polymers and nanoparticulate systems on insulin absorption in Caco-2 cells, Eur. J. Pharm. Biopharm. 70 (2008) 270–278. [186] Y. Zambito, C. Zaino, G. Di Colo, Effects of N-trimethylchitosan on transcellular and paracellular transcorneal drug transport, Eur. J. Pharm. Biopharm. 64 (2006) 16–25. 117 [187] A. Enriquez de Salamanca, Y. Diebold, M. Calonge, C. Garcia-Vazquez, S. Callejo, A. Vila, M.J. Alonso, Chitosan nanoparticles as a potential drug delivery system for the ocular surface: toxicity, uptake mechanism and in vivo tolerance, Invest. Ophthalmol. Vis. Sci. 47 (2006) 1416–1425. [188] N. Forsberg, A. Von Malmborg, K. Madsen, W. Rolfsen, S. Gustafson, Receptors for hyaluronan on corneal endothelial cells, Exp. Eye Res. 59 (1994) 689–696. [189] S.N. Zhu, B. Nolle, G. Duncker, Expression of adhesion molecule CD44 on human corneas, Br. J. Ophthalmol. 81 (1997) 80–84. [190] L. Collis, C. Hall, L. Lange, M. Ziebell, R. Prestwich, E.A. Turley, Rapid hyaluronan uptake is associated with enhanced motility: implications for an intracellular mode of action, FEBS Lett. 440 (1998) 444–449. [191] L. Contreras-Ruiz, M. De la Fuente, C. García-Vázquez, V. Sáez, B. Seijo, M.J. Alonso, M. Calonge, Y. Diebold, Improved in vivo tolerance of hyaluronic acid chitosan nanoparticles, Cornea, In press (MS No ICO201509). [192] D.F. Williams, On the mechanisms of biocompatibility, Biomaterials 29 (2008) 2941–2953. [193] S. Hirano, H. Seino, Y. Akiyama, I. Nonaka, Biocompatibility of chitosan by oral and intravenous administration, Polym. Eng. Sci. 59 (1989) 897–901. [194] E. Barbu, L. Verestiuc, T.G. Nevell, J. Tsibouklis, Polymeric materials for ophthalmic drug delivery: trends and perspectives, J. Mater. Chem. 16 (2006) 3439–3443. [195] J.A. Gomes, R. Amankwah, A. Powell-Richards, H.S. Dua, Sodium hyaluronate (hyaluronic acid) promotes migration of human corneal epithelial cells in vitro, Br. J. Ophthalmol. 88 (2004) 821–825. [196] G. Camillieri, C. Bucolo, S. Rossi, F. Drago, Hyaluronan-induced stimulation of corneal wound healing is a pure pharmacological effect, J. Ocul. Pharmacol. Ther. 20 (2004) 548–553. [197] A. Genasetti, D. Vigetti, M. Viola, E. Karousou, P. Moretto, M. Rizzi, B. Bartolini, M. Clerici, F. Pallotti, G. De Luca, A. Passi, Hyaluronan and human endothelial cell behavior, Connect. Tissue Res. 49 (2008) 120–123. [198] E.A. Balazs, M.I. Freeman, R. Kloti, G. Meyer-Schwickerath, F. Regnault, D.B. Sweeney, Hyaluronic acid and replacement of vitreous and aqueous humor, Mod. Probl. Ophthalmol. 10 (1972) 3–21. [199] M.E. Johnson, P.J. Murphy, M. Boulton, Effectiveness of sodium hyaluronate eyedrops in the treatment of dry eye, Graefes Arch. Clin. Exp. Ophthalmol. 244 (2006) 109–112. [200] P.A. Knepper, A.I. Farbman, A.G. Telser, Exogenous hyaluronidases and degradation of hyaluronic acid in the rabbit eye, Invest. Ophthalmol. Vis. Sci. 25 (1984) 286–293. [201] D.M. Schwartz, M.D. Jumper, G.M. Lui, S. Dang, S. Schuster, R. Stern, Corneal endothelial hyaluronidase: a role in anterior chamber hyaluronic acid catabolism, Cornea 16 (1997) 188–191. [202] S.H. Pangburn, P.V. Trescony, J. Heller, Lysozyme degradation of partially deacetylated chitin, its films and hydrogels, Biomaterials 3 (1982) 105–108. [203] K. Tomihata, Y. Ikada, In vitro and in vivo degradation of films of chitin and its deacetylated derivatives, Biomaterials 18 (1997) 567–575.