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Applications of nanoparticles in ophthalmology

Yolanda Diebold a,b,*, Margarita Calonge a,b

a Ocular Surface Research Group, Edi ficio IOBA, Campus Miguel Delibes, Instituto de Oftalmobiología Aplicada (IOBA), Universidad de Valladolid, Paseo de Belén, 17,

E-47011 Valladolid, Spainb CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Spain

Keywords:

Drug delivery

Gene delivery

Nanomedicine

Nanoparticles

Ocular drug administration

Toxicity

a b s t r a c t

Nanocarriers, such as nanoparticles, have the capacity to deliver ocular drugs to specific target sites and

hold promise to revolutionize the therapy of many eye diseases. Results to date strongly suggest thatocular medicine will benefit enormously from the use of this nanometric scale technology. One of the

most important handicaps of the eye as a target organ for drugs is the presence of several barriers thatimpede direct and systemic drug access to the specific site of action. Superficial barriers include

the ocular surface epithelium and the tear film, and internal barriers include the bloodeaqueous andblooderetina barriers. Topical application is the preferred route for most drugs, even when the target

tissues are at the back part of the eye where intraocular injections are currently the most common routeof administration. Direct administration using any of these two routes faces many problems related to

drug bioavailability, including side effects and repeated uncomfortable treatments to achieve therapeuticdrug levels. In this regard, the advantages of using nanoparticles include improved topical passage of

large, poorly water-soluble molecules such as glucocorticoid drugs or cyclosporine for immune-related,vision-threatening diseases. Other large and unstable molecules, such as nucleic acids, delivered using

nanoparticles offer promising results for gene transfer therapy in severe retinal diseases. Also,nanoparticle-mediated drug delivery increases the contact time of the administered drug with its targettissue, such as in the case of brimonidine, one of the standard treatments for glaucoma, or corticosteroids

used to treat autoimmune uveitis, a severe intraocular inflammatory process. In addition, nanocarrierspermit the non-steroidal anti-inflammatory drug indomethacin to reach inner eye structures using the

transmucosal route. Finally, nanoparticles allow the possibility of targeted delivery to reach specific typesof cancer, such as melanoma, leaving normal cells untouched.

This review summarizes experimental results from our group and others since the beginnings of nanocarrier technology to deliver drugs to different locations in the eye. Also, it explores the future

possibilities of nanoparticles not only as drug delivery systems but also as aides for diagnostic purposes.Ó 2010 Elsevier Ltd. All rights reserved.

Contents

1. Introduction: what is nanomedicine? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5972. Drug delivery systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .597

3. The eye as a target organ for drug delivery systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .597

4. Types of NPs for ocular delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599

5. NPs and the anterior segment of the eye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 600

6. NPs and the posterior segment of the eye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 603

Abbreviations: AS-ODNs, Antisense oligonucleotides; CS, Chitosan; CSO, Chitosan oligomers; CsA, Cyclosporine A; ESF, European Science Foundation; HA, Hyaluronic acid;

HA-CS NPs, Hyaluronic acid and chitosan-based nanoparticles; HA-PECL NPs, Hyaluronic acid-coated poly- e-caprolactone nanoparticles; IOP, Intraocular pressure; LCS-NPs,

Liposomeechitosan nanoparticle complexes; NIH, National Institutes of Health; NPs, Nanoparticles; OIR, Oxygen-induced retinopathy; PBCA, Poly(butyl-cyanoacrylate);

PECL, Poly-e-caprolactone; PEG, Polyethyleneglycol; pGFP, plasmid green fluorescent protein; PIBCA, Poly(isobutyl-cyanoacrylate); PLA, Poly-D-lactic acid; PLGA, Poly-D-

lactic-co-glycolide; RNAi, RNA interference; rAAV, adeno-associated virus vectors; RPE, Retina pigment epithelium; siRNA, small interfering RNA.

* Corresponding author. Ocular Surface Research Group, Edificio IOBA, Campus Miguel Delibes, Instituto de Oftalmobiología Aplicada (IOBA), Universidad de Valladolid,

Paseo de Belén, 17, E-47011 Valladolid, Spain. Tel.: þ34 983 184750; fax: þ34 983 184762.

E-mail address: [email protected] (Y. Diebold).

Contents lists available at ScienceDirect

Progress in Retinal and Eye Research

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 om / l o c a t e / p r e r

1350-9462/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.

doi:10.1016/j.preteyeres.2010.08.002

Progress in Retinal and Eye Research 29 (2010) 596e609



7. NPs and gene delivery/therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 604

8. Nanoparticle safety: toxicity and interaction with the immune system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 605

9. Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607

10. Summary and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607

11. Author disclosure statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607

1. Introduction: what is nanomedicine?

In 2003, theEuropeanScienceFoundation (ESF)initiated a projectaimed at gatheringEuropeanexpertsfrom academia andindustry to

prepare what was called ‘ESF Forward Look on Nanomedicine’,published in 2004 (http://www.esf.org/publications/forward-looks.html). Several workshops were conducted to (i) define the field,(ii) discussthe future impactof nanomedicine on healthcare practice

and society, (iii) review the state-of-the-art of research, (iv) identify

Europe’s strengths and weaknesses, and (v) deliver recommenda-tions on research trends and organization. These experts formallydefined the field of ‘nanomedicine’ as “the science and technology

of diagnosing, treating and preventing disease and traumatic injury,of relieving pain, and of preserving and improving human health,usingmoleculartools and molecularknowledge of the humanbody”.It is noteworthy that this concept is based in complex systems of

nanometre-scale size, i.e., from one nanometre to hundreds of nanometres, with theultimategoal of using them to achieve medicalbenefits. It is important to bear in mind that the nanometre scale isthe scale at which molecules and compounds operate inside living

cells.Soon afterward the ESF’s publication, the National Institutes of

Health (NIH) in the U.S.A. developed a Nanomedicine Roadmap

Initiative (http://nihroadmap.nih.gov/nanomedicine/). As a centre-piece of this initiative, a national network of eight collaborativeNanomedicine Development Centers was established in 2006. Thatmultidisciplinary research initiative was primarily directed towards

gathering extensive information about nanoscale intracellularbiological structures. That information was to be used in theapplication of newly developed nanomedical therapies to treatspecific diseases. As an example of the interest that this topic has

awakened in the vision research community, an education course

‘Nanotechnology and Nanomedicine: Applications for VisionResearch’, was organized in 2005, co-sponsored by the Associationfor Research in Vision and Ophthalmology and the NIH ’s National

Eye Institute.As anyonecan envision, nanomedicine has a relevant position in

the global agenda for future development of medical research inthe 21st Century. One of the main topics in nanomedicine research

is the pharmaceutical development of drug delivery systems. Itsgoal is the development of improved nano-sized drug carriersconsisting of at least two components, one of which is the activetherapeutic ingredient. These drug-loaded carriers can be termed

‘nanopharmaceuticals’ or ‘nanomedicines’ in a broad sense. Thisreview will focus on the ocular applications of nanoparticles (NPs),a particular type of these drug delivery systems.

2. Drug delivery systems

Among the different approaches that have been taken todevelop more ef ficient treatments to fight against human and

animal life-threatening or debilitating diseases, the development of drug delivery systems is noteworthy. The purpose of a drug deliverysystem is to act as a carrier or vehicle for an entrapped or bound

therapeutic agent to reach precisely and effectively the desired siteof action. Here we focus on delivery systems that target ocularstructures. This concept is particularly interesting when one takesinto account the physicochemical features of the frequently mar-

keted biotechnological macromolecules, such as peptides, protein,antibodies, and nucleic acids (Conti et al., 2000; Degim and Celebi,2007; Levy-Nissenbaum et al., 2008).

However, a drug delivery system is more than a simple (nano)

carrier. All of the science and technology behind the design of drug

delivery systems intends to achieve solutions for key aspects of modern treatments: (i) to control the release of the active agent sothat a therapeutic concentration is maintained over a prolonged

period of time, (ii) to develop organ or site-specific or even disease-specific targeting, and (iii) to provide new or more convenientroutes of administration for drugs able to reach those locations inthe body that are dif ficult to access. The ultimate goals are to better

manage relevant drug-related parameters, such as pharmacoki-netics, pharmacodynamics, non-specific toxicity, immunogenicity,biorecognition, and to improve therapeutic ef ficacy (Vanderwootand Ludwig, 2007; Sahoo et al., 2008).

There are different kinds of drug delivery technologies that aredesigned to serve as drug delivery systems (Medina et al., 2007;Sahoo et al., 2008; Gaudana et al., 2009). These include, among

others, transdermal patches, implants, nanodevices, and cellencapsulation devices. Among nanoparticulate-based drug deliverysystems (or nanosystems) (Table1) one can find different polymericformulations made of non-degradable polymers and biodegradable

polymers that are either hydrophilic or hydrophobic. Examplesof nanosystems that differ in composition include the following:(i) Nanoparticles (NPs) consist of 1 mm or smaller particlescomposed of various polymers or materials. These are described in

detailbelow.(ii) Liposomes are lipidicmembranes, similarto plasmamembranes, and surround an aqueous core. A variant of liposomesare niosomes, consisting of non-ionic surfactants. (iii) Emulsionsconsist of stabilised oil-in-water or water-in-oil mixtures. Others

include (iv) nanosuspensions, (v) dendrimers, (vi) nanoparticle-loaded contact lenses, (vii) nanotubes and fullerenes composed of

carbon-based nanomaterials, and (viii) quantum dots made of semiconductor materials with fluorescent properties and covered

with other materials. However, drug delivery systems other thanthose collectively named as “nanoparticles” are out of the scope of this review, and therefore we will not comment on them.

3. The eye as a target organ for drug delivery systems

There are a plethora of ocular disorders that may be vision-threatening. The responsiveness towards classically developeddrugs is limited and most fail to correct the underlying problem.Thus, there is a scarcity of truly curative treatments for most eye

diseases. The main reasons for these limitations are bio-pharmaceutical problems related to the special characteristic of the

eye that restricts drug bioavailability. The eye is partially isolatedfrom the remainder of the body by several types of barriers that

impede the effective passage of many drugs (Fig. 1), leading to

Y. Diebold, M. Calonge / Progress in Retinal and Eye Research 29 (2010) 596 e609 597



a minimal dose absorption (Urtti, 2006). These barriers consist of the (i) muco-aqueous layer of the tear film that protects the ante-

rior surface of the eye, (ii) corneal epithelium with abundant tight junctions and desmosomes, (iii) iris blood vessels that lack fenes-trations, (iv) non-pigmented layer of the ciliary epithelium thatconstitutes the bloodeaqueous barrier and limits the passage of

molecules from the blood to the inner part of the eye, and (v) retinalpigment epithelium (RPE), along with the endothelium of theretinal vessels, constitute the inner and the outer blooderetinabarriers, respectively, that limit the passage of molecules from the

blood to the retina and vitreous cavity.Additionally, certain physiological processes contribute to the

poor ef ficacy of conventional drug formulations. For instance,blinking and tear drainage through the lachrymal drainage system

act to reduce the residence time of topically applied molecules. Eyedrops placed onto the ocular surface are washed away in less than

30 s (Kaur and Kanwar, 2002). The maintenance of corneal trans-parency is based upon several strategies, one of them being the

sealing of the corneal epithelium by means of specialized struc-tures, such as tight junctions and desmosomes. The cornealepithelium is therefore almost impermeable to any substancelarger than 500 Da (Hämäläinen et al., 1997). Most of the commonly

used topical drugs are larger than that and, consequently, do notcross the cornea. Instead, they permeate throughout the conjunc-tiva and the underlying sclera in what is known as “non-productivepassage”. Altogether, less than 5% of topically administered drugs

reach intraocular tissues (Keister et al., 1991). Therefore, the exis-tence of several ocular tissue and cell barriers along with thephysiological processes impede the effective passage of manydrugs, leading to a minimum dose absorption into the eye.

Drug delivery systems hold promise to be an important part of the new therapeutic armamentarium in ophthalmology. Since theearly study of Wood et al. (1985) showing the intrinsic capacity of NPs to adhere to the ocular surface and interact with the epithe-

lium, applications of nanotechnologies to solve eye problems havebeen sought. Knowledge derived from drug delivery systems usingnon-ocular routes of administration has stimulated researchers to

find applications for them in ophthalmology. What makes them

attractive to treat eye diseases is the possibility of the controlledrelease of drugs, especially poorly water-soluble ones, thatsurpasses the ocular barriers and effectively reaches the target.Nanoparticulate systems improve the delivery of poorly water-

soluble drugs while significantly reducing toxicity compared to thefree drug. Increasing attention has been focused particularly on thisaspect due to the clear therapeutic implications. Also, the micro- ornano-size of such drug carriers is appealing.

To envision the potential of nanoparticles as drug carriers that

can treat ocular disorders, it is necessary to understand thepeculiarities of the drug administration routes in the eye. First,local delivery of the drug is preferred over systemic delivery. There

are several different modalities for ocular drug administration(Table 2). The most common include liquids topically applied ontothe front of the eye in the form of eye-drops, sub-conjunctivalor sub-Tenon’s injection in the conjunctival tissue or below

the Tenon’s capsule, and intravitreal injection. Also, intraocularimplants made of either biodegradable or non-biodegradablepolymers loaded with different drugs are used to provide long-

term drug presence at the implantation site. Topical administra-tion of drugs is used to alleviate the symptoms and signs causedby ocular surface inflammatory disorders, such as dry eyesyndrome and allergic diseases that affect millions of people

worldwide. They are also used to treat infections and complex,vision-threatening diseases, such as glaucoma or intraocularinflammation (uveitis). In most cases, the patients themselves

Fig. 1. Schematic presentation of the ocular structure showing a summary of ocular

pharmacokinetics. The numbers refer to following processes: 1) transcorneal perme-

ation from the lachrymal fluid into the anterior chamber, 2) non-corneal drug

permeation across the conjunctiva and sclera into the anterior uvea, 3) drug distri-

bution from the bloodstream via the bloodeaqueous barrier into the anterior chamber,

4) elimination of drug from the anterior chamber by aqueous humour passage into the

trabecular meshwork and Sclemm’s canal, 5) drug elimination from the aqueous

humor into the systemic circulation across the bloodeaqueous barrier, 6) drug

distribution from the blood into the posterior eye across the blooderetina barrier, 7)

intravitreal drug administration, 8) drug elimination from the vitreous via the poste-

rior route across the blooderetina barrier, and 9) drug elimination from the vitreous

via the anterior route to the posterior chamber. (Taken from Urtti A., Adv Drug Deliv

Revs (2006), with permission of Elsevier)

Table 1

Nanoparticulate drug delivery systems (or nanosystems) used as carriers for drug administration.

Nanosystem Composition Potential application

in the eye

Nanoparticles Natural or synthetic polymers, metals, lipids, phospholipids Yes

Liposomes Phospholipids Yes

Niosomes Non-ionic surfactants Yes

Emulsions Oil-in-water and water-in-oil mixtures that require surfactants Yes

Nanosuspensions Inert polymer resins Yes

Dendrimers Synthetic polymers Yes

Nanoparticle-loaded contact lenses Different hydrogel-based lenses with nanoparticulate-based

drugs incorporated in the lens matrix

Yes

Nanotubes and fullerenes Carbon-based nanomaterials Not tested yet

Quantum dots Semiconductor materials covered with other materials Yes (diagnostic)

According to Sahoo et al. (2007) and Gaudana et al. (2009).

Y. Diebold, M. Calonge / Progress in Retinal and Eye Research 29 (2010) 596 e609598



instil the drops many times a day, and the treatment is not usually

curative but just palliative.To summarize the necessity of using a carrier for ocular

drugs, the key is to achieve adequate bioavailability. There aremany challenges for ocular drug delivery systems. In some cases,

the goal is to stop or reverse problems such as degenerations inthe retina and neovascularisation in the cornea and in the retina.In other cases, the drugs must contribute to the success of refractive corneal surgery and the healing process, tissue

transplantation and growth factor delivery for retinal andcorneal regenerative medicine, and gene therapy for hereditaryretinal disorders.

4. Types of NPs for ocular delivery

There has been an evolution in the design of nanoparticles(NPs), and Sánchez and Alonso (2006) extensively reviewed thefeatures of polymers used to prepare the carriers. We analyze in

this review the progress that has been made from a practical pointof view in terms of therapeutic and diagnostic applications of NPs

in the field of ophthalmology. The general term ‘nanoparticle’ willbe used in this review for the sake of simplification. However,this term can refer to nanospheres or nanocapsules, all of whichare more properly designated as colloidal systems (Fig. 2). Nano-

spheres are matrical-type nanostructures that entrap or adsorb the

biologically active molecule onto the surface. Nanocapsules arereservoir-type nanostructures within a surrounding polymeric wallcontaining an oil core where the active molecule is dissolved. These

nanostructures can be coated with a hydrophilic polymer or evenfunctionalized with antibodies attached to the coating.

There are different methods to prepare NPs and load them withtherapeutic molecules (for review, please see Pinto-Reis et al.,

2006). NPs can be made of a great variety of materials includingorganic carbon-based biopolymers and inorganic ceramic, metallic,and semiconductor materials. Some of the most commonly usedbiomaterials include polyacrylates, polyalkylcyanoacrylates, poly-

lactide (PLA), polylactideepolyglycolide (PLGA), polycaprolactones,dextran, albumin, gelatin, alginate, collagen, hyaluronic acid, andchitosan. The possibilities for their design are almost infinite. The

intended application infl

uences the kind of material used for theirpreparation. The different chemical ways in which bioactivemolecules can be associated with polymers and give rise to drug-loaded NPs include entrapment, encapsulation, adsorption, or

attachment to a polymer.Also, NPs can be prepared in different sizes, charge, and other

physicochemical features. This confers great versatility upon them.The physicochemical characteristics of NPs not only confer versa-

tility in terms of the kind of drug to be loaded, but they also influ-ence the cellular uptake and intracellular traf ficking. Additionally,the physicochemical characteristics are critical for other properties,such as interaction with plasma proteins, other blood components

(Dobrovolskaia et al., 2008), and with immune cells (Dobrovolskaiaand McNeil, 2007), all of which are relevant to the organ distribu-tion. For instance, opsonisation of NPs covers the surface by opso-nins present in the blood and creates a “molecular signature”(Aggarwal et al., 2009) that determines the route of NP internali-zation in phagocytic cells and eventually their fate. However, if theNP surface is covered by a hydrophilic coat of poly(ethylene glycol)(PEG), opsonisation is prevented. This has been termed a ‘stealth

effect’ (Gref et al., 1994), and it reduces the rate of cellular uptake.Consequently, PEG-NPs stay longer in the bloodstream. In addition,the rate of clearance of NPs is an important consideration withregard to potential toxicitycausedby their permanence in thetarget

tissue.Therapeutic targets for drugs can be located extra- or intra-

cellularly. With regard to classical drugs with sizes greater than

Fig. 2. Evolution of colloidal systems: The first generation of colloidal systems consisted of polymerized matrices known as nanoparticles and nanocapsules composed of

a polymeric wall containing an oil core. Second generation systems were similar to nanocapsules, but with improved hydrophilicity associated with a coating polymer. Third

generation systems are functionalized by the attachment of antibodies or lectins, among other targeting moieties to the surface structure. (Adapted from Sánchez A. and Alonso M.J.,

2006).

Table 2

Modalities for ocular drug administration.

Modality Anatomical location Kind of

administered drug

Topical eye-drops Ocular surface

(onto the tear film and

corneal and conjunctival

epithelia)

Artificial tearsAnti-infectives

Anti-allergics

Anti-inflammatories

Anti-hypertensives

Anaesthetics

Mydriatics, miotics,

cyclopegics

Periocular injection

Sub-conjunctival Sub-conjunctval space Anti-infectives

Mydriatics

Corticoids

Sub-Tenon

Transeptal Anterior orbit Anaesthetics

Corticoids

Retro-orbital Posterior orbit Anaesthetics

Corticoids

Intraocular injection

Intracameral Anterior chamber Anti-infectivesIntravitreal Vitreous Anti-infectives

Anti-angiogenics

Corticoids




1 kDa, stability conditions or hydrophilicity makes them unable tocross plasma membrane. NPs, as well as other drug deliverysystems, enable the loaded bioactive molecule to cross cellmembranes and epithelial barriers by using different internaliza-

tion pathways. This is one of the most interesting aspects of this

technology. Reports showing that submicrometer delivery systemssuch as NPs can enter cells and become concentrated into non-diffusible molecules appeared in the 1970s (Couvreur et al., 1977).

Much research has been done since then and much knowledge hasbeen gained related to both carrier design and mechanisms of biological action. It is now known that the physicochemical char-acteristics of nanocarriers, such as size, shape, surface charge,

surface coating, and surface functionalization with targetingligands, along with the target cell type, determine the internali-zation pathway and the intracellular fate.

Increasing numbers of studies about cell targeting and internali-

zation pathways are being accomplished, mainly using nano-medicines to treat infectious diseases and cancers. Recent interestingreviews deal with these topics (Bareford and Swaan, 2007; Frenkel,

2008; Hillaireau and Couvreur, 2009). The main internalizationpathways for NPs discussed in those reviews are summarized in Table3. Phagocytosis is the typical pathway for NPs intravenously admin-istered. It implies NP opsonisation as mentioned above and the

involvement of specific cells with phagocytic capacity that will ingestthe NPs. In the cytoplasm, NPs and their payload will becomeaccessible to lysosomes and experience enzymatic degradation. Thisstep is important to ensure drug release.

Endocytosis occurs in all mammalian cells by means of pitted orinvaginated membrane regions that are coated by either of twospecific proteins. Pitted membranes coated by clathrin can beformed by receptor-mediated and receptor-independent processes

with different internalization rates. Invaginated membranes coatedby another protein, caveolin, can also conduct receptor-mediatedendocytosis. NPs can be targeted to interact with cell receptors of interest to facilitate internalization by either of these receptor-

mediated endocytic mechanisms. Loaded NPs internalized by cla-thrin-mediated endocytosis are directed to lysosomal degradation.Those internalized by caveolin-mediated endocytosis accumulatein the endosomes (caveosomes) and are delivered to other

subcellular compartments different from lysosomes. This lastmechanism has been used, for instance, to deliver chemothera-peutics to cancer cells. Also, clathrin- and caveolin-independentendocytosis mechanisms have been described.

Finally, macropynocytosis also occurs in all kind of cells. It isdirected by actin-driven membrane protrusions that form large(>1 mm) endocytic vesicles (macropinosomes). Eventually, thosevesicles fusewith lysosomes. This pathway is the least specific of allmentioned. Notably, several endocytic mechanisms can take place

simultaneously. The above mentioned review articles presentabundant examples of all these mechanisms for different kinds of NPs tested in a variety of cells and tissues. Surprisingly, there isa scarcity of studies about internalization pathways and intracel-

lular traf ficking of NPs intended for ocular application (see Table 1).

Intracellular metabolism of delivered drugs differs according tothe internalization pathway. It implies a physical separation fromthe NP transporter, and their physicochemical characteristics will

determine the degradation ratio. These are key aspects for thepharmacological activity of the carried molecule. Different kineticprocesses are described to control the release profiles of drugs fromNPs,including (i) desorption of the surfacebound or adsorbed drug,

(ii) diffusion through the NP matrix or the polymer wall, (iii) NPwall erosion, and (iv) a combination of erosion and diffusionprocesses (Soppimath et al., 2001; Harush-Frenkel et al., 2008). Fordrugs that uniformly distribute or dissolve through the NP matrix,

diffusion and biodegradation govern the release process. If drugdiffusion is faster than polymeric degradation, drug release mainlyoccurs through diffusion; if not, then drug release will occurs

through degradation of the polymer.Finally, there are other important factors involved in the designof a nanomedicine,including themodality of administration suchasinjection or topical application, and the features of the entrapped

drug itself. This complex picture is completed with the requirementof minimal potential toxicity (see specific chapter) and the contin-uous need for improved ef ficiency. Different kinds of NPs preparedwith some of the materials described above have been studied in

the search for alternatives in ophthalmic treatments. We present inthis review those that have been tested either in vitro or in vivo.

5. NPs and the anterior segment of the eye

The bioavailability of an instilled conventional drug onto theocular surface is usually low. Most of it is lost due to physiological

mechanisms, such as tear drainage and blinking, a few secondsafter instillation (Bayens and Gurny, 1997). Thus, there is a shortpre-corneal residence time, usually less than 2 min, and a non-productive absorption thorough the well vascularised conjunctiva

and the nasolachrymal drainage system ( Jarvinen et al., 1995).Therefore, the picture we face includes a very limited absorption of drug, a potential although limited access to intraocular tissuesthrough the conjunctivalescleral pathway, and the risk of systemic

side effects. For those reasons, intensive research in recent decadeshas focused on increasing the corneal penetration of topicallyapplied drugs (Schoenwald, 1990; Sasaki et al., 1999; Edwards andPrausnitz, 2001; Mannermaa et al., 2006). NPs were considered to

offer the possibility of a more facile delivery and transport acrosstissues, and consequently their potential started to be studied.

Table 3

Main pathways for nanoparticle (NP) internalization and preferential degradation route.

Pathway Target cells Endocytic

vesicles size

Degradation route Examples of NPs

studied for

ocular delivery

Phagocytosis Professional phagocytes 0.25e10 mm Early endosomes and lysosomes e

Clathrin-mediated endocytosis All mammalian cells z120 nm Early endosomes and lysosomes SLNs loaded with pCMS-EGFP

(del Pozo-Rodríguez et al., 2008)

Caveolin-mediated endocytosis All mammalian cells z60 nm Endosomal accumulation;

nondegradative pathway

HAS NPs loaded with SOD1 gene

(Mo et al., 2007)

HA-CSO NPs loaded with pSEAP

(Contreras-Ruiz et al., Submitted for publication)

Clathrin- and caveolin-independent

endocytosis

All mammalian cells z90 nm Early endosomes PLGA NPs loaded with 6-cumarin

(Qaddoumi et al., 2003)

Micropynocytosis All mammalian cells >1 mm Macropinosomes and lysosomese

SLNs, solid lipidNPs; HSANPs, human serum albumin NPs;SOD1, Cu, Zn superoxide dismutasegene; pCMS-EGFP, plasmid DNA encodingenhanced greenfluorescent protein;

HA-CSO, hyaluronic acid-chitosan oligomers; pSEAP, plasmid DNA encoding secreted alkaline phosphatise; PLGA, Poly- D-lactic-co-glycolide.




In 1986, an early attempt to use NPs was done with poly(butyl-cyanoacrylate) (PBCA) NPs loaded with progesterone, a highlylipophilic molecule (Li et al., 1986). The NPs were topically appliedto rabbit eyes and concentrationetime profiles in different ocular

tissues were assessed. The authors found a decreased progesterone

concentration in tissues when compared to control solutions,suggesting a greater af finity of progesterone for the NP polymerthan expected. As a consequence, progesterone was less available

for absorption during its residence time in the pre-corneal area. Thelesson learned from these experiments was that it is important tooptimize the physicochemical relations between the polymers anddrug to obtain an ef ficient carrier. Later, Losa et al. (1993), usedpoly

(isobutyl-cyanoacrylate) (PIBCA) and poly-e-caprolactone (PECL)nanocapsules for the ocular delivery of metipranolol, a beta-blockerused to treat certain types of glaucoma. In that work, the polymercoating was not responsible for the differences observed regarding

the controlled release of the encapsulated drug. Instead, the drugrelease profile was mainly influenced by the type of oil core in thenanocapsule. However, a certain contribution of the polymer

coating on emulsion stability was acknowledged. Our group laterstudied PECL nanocapsules loaded with 1% cyclosporine (CsA) ina rat model of corneal transplantation rejection ( Juberías et al.,1998). This formulation had been developed to improve the

corneal penetration of CsA applied topically (Calvo et al.,1996). Thiswell-known immunosuppressive drug, widely used in trans-plantation patients, has severe systemic side effects such as neph-rotoxicity, hepatotoxicity, and hypertension. These side effects have

limited its use in the local management of immune rejection of corneal grafts. By using PECL nanocapsules, toxic effects of systemicadministration of CsA were avoided along with improved ocularpenetration. While corneal graft rejection was not prevented by the

CsA-loaded PECL NPs, the failure was not considered to bea consequence of negative interactions between the polymer andthe drug.

Improved formulations using polymers with known biocom-

patibility and biodegradability, such as poly-D-lactic acid (PLA) andits copolymer glycolic acid (PLGA), were developed later. Flurbi-

profen-loaded PLGA NPs successfully reduced inflammation in anin vivo rabbit model of ocular inflammation (Vega et al., 2006). Incomparison with commercial flurbiprofen formulations, flurbipro-fen-loaded NPs were more effective in reducing inflammation as

evaluated by direct observation of clinical signs.More recently, we and others have developed applications using

chitosan (CS)-based nanosystems. CS is a natural polysaccharidewith interesting features, such as biocompatibility and biodegrad-

ability, mucoadhesiveness, and the ability to transiently enhancethe permeability of mucosal barriers. These features have made itquite useful in the development of drug delivery systems for

transmucosal administration (for review see Alonso and Sánchez,2003; Paolicelli et al., 2009). NPs made of CS and carbopol,a cross-linked polymer of acrylic acid (Kao et al., 2006), combinedproperties of both polymers, such as stability in aqueous solution,small size, improved biocompatibility, and positive charge to

facilitate interaction with the negatively charged biologicalmembranes. CS/carbopol NPs can be loaded with pilocarpine,a parasympathomimetic drug used as treatment for open-angle

glaucoma. Patients with this form of glaucoma need to frequentlyinstil pilocarpine eye-drops, which increases aqueous humouroutflow for only 4e8 h Kao et al. (2006) compared the ef ficacy of pilocarpine-loaded NPs against liposomes, gel, and eye-drops

formulations of pilocarpine in normal rabbits. Pilocarpine inducesa miotic response measured as a decrease in pupil diameter, and the

effect of the CS/carbopol NPs lasted up to 24 h, much longer thanthe other formulations. The release profile, previously studied in

vitro, showed an initial burst release followed by a sustained

release for at least 24 h. These results are very promising, andfurther evaluation in a glaucoma animal model is now warranted.Recently, another CS-based nanosystem, CS/sodium alginate NPs,

has been reported as a potential reservoir for topical delivery of gatifloxacin, a potent fourth-generation fluoroquinolone with low

intraestromal penetration that is mainly used for microbial keratitis(Motwani et al., 2008), but so far, the NP characterization has been

only physicochemical, and no in vitro or in vivo experiments havebeen reported.

The transport pathways by which NPs penetrate the ocularsurface tissues are of great interest. Zimmeret al. (1991) studied theocular transport pathway of fluorescein-labeled PBCA NPs in

rabbits. They found the fluorescence signal localized insideconjunctival and corneal epithelial cells, and observed differencesin depth of tissue penetration. They proposed a variety of pathwaysto explain their data, including NP endocytosis, lysis of cell

membrane by NP metabolic degradation products, and a trans-cellular route. The transcellular route was also proposed for coatedPECL nanocapsules (de Campos et al., 2003).

A critical question with regard to NP delivery of drugs is theconcentration and duration of the drug in the target andsurrounding tissues. Losa et al. (1991) tested PBCA NPs withdifferent stabilizer agents to improve the binding of the antibiotic

amikacin sulphate to the NPs. One of the formulations, usingDextran 70000 as a stabilizer, resulted in a significant increase of the amikacin concentration not only in the cornea but also in theaqueous humour. de Campos et al. (2001) studied the distribution

of CsA-loaded CS NPs in different rabbit ocular tissues. In that study,therapeutic concentrations of this immunosuppressant drugadequate to modulate the local immune response were maintainedin the cornea and conjunctiva for 48 h post-administration.However, those concentrations were not achieved using a formu-

lation consisting on a CsA suspension in either a chitosan aqueoussolution or a CsA suspension in water. The concentration of CsA inaqueous humour, iris, and ciliary body were extremely low. Inaddition, no detectable CsA levels were measured in plasma.

Therefore, a prolonged local drug delivery was achieved using theCS NPs with no significant accumulation in intraocular tissues.

The surface characteristics of the nanocarriers also have aninfluence on the interaction with the ocular surface structures. For

instance, CS-coated PECL nanocapsules enter the corneal epithe-lium in vivo more ef ficiently than uncoated PECL or polyethyleneglycol-coated PECL nanocapsules (de Campos et al., 2003). We havealready mentioned the exceptional biological features of CS, espe-

cially mucoadhesiveness and the ability to transiently enhance thepermeability of mucosal barriers. Our group is most interested inthis approach using mucoadhesive NPs able to reach the anteriorstructures of the eye after topical administration. Westarted testing

CS NPs because of the great potential envisioned for this polymer inthe ophthalmology field (Alonso and Sánchez, 2003). We reportedthat CS NPs did not cause toxicity-related alterations in several celllines derived from human conjunctiva epithelium (de Campos

et al., 2004; Enríquez-de-Salamanca et al., 2006). We were able toidentify albumin-loaded CS NPs inside the cells by fluorescencemicroscopy in a time-dependent manner. With in vivo experimentsusing albino rabbits, we confirmed that CS NPs were well-tolerated

by ocular surface structures, causing no harm or inflammation, asdetermined by histopathological analysis. The corneal andconjunctival tissues that took up the CS NPs showed interestingtissue-related distribution patterns (Fig. 3). Both cornea and

conjunctiva incorporated the NPs; however, the conjunctiva wasmore permeable to the nanocarrier as shown by the deeper pene-

tration into the epithelium and underlying stroma. Other authorshave also explored similar strategies. For instance, Yenice et al.

(2008) used hyaluronic acid (HA)-coated PECL nanospheres for




corneal CsA delivery. The concentrations in rabbit cornea were10e15-fold higher than that achieved when CsA was administered

as solution in castor oil.These results moved us to explore different biomaterial

combinations intended specifically for the ocular surface tissues.Consequently we joined liposomes and CS NPs to form a newnanosystem that we termed “liposomeechitosan nanoparticles”(LCS-NPs) (Diebold et al., 2007). The theoretical advantages of thesecomplexes are the combination liposome biocompatibility withbiological membranes and the demonstrated properties of CS NPs.

We tested three different complex formulations, showing againtheir potential for ocular administration. Using mucus-producingprimary cultures of conjunctival epithelium, we observed that thethree nanosystems were first retained in the in vitro mucus layer

and then entered the epithelial cells, depending on the particular

NP composition (Fig. 4). We consider this feature a potentialadvantage that can be used to modulate the retention time of thenanocarrier in the tear film. Different in vivo retention times wouldbe required depending on the encapsulated drug.

Use of different lipid carriers has had renewed interest. Anexample is the recent paper by Attama et al. (2009) that presentsphospholipid nanoparticles made in theobroma oil. These weredesigned to incorporate timolol hydrogen maleate, a water-soluble

drug used as a first-choice treatment for glaucoma. Using a modi-fied Franz diffusion cell apparatus, they did drug permeationexperiments using a bio-engineered cornea construct, composed of human immortalized corneal endothelial cells, stromal fibroblasts,

and epithelial cells. This in vitro study, although showing promising

Fig. 4. Confocal microscopy images of primary cultures of human conjunctival epithelium. Control cultures had normal morphology when viewed with transmitted light (TL).

During 30 min of incubation, LCS-NP complexes (green) passed through the mucus layer and were present in deeper cell layers. They formed aggregates with different patterns for

each type of LCS-NP tested. Z-series profile images (lowest panels) are projections of stacked image profiles from optical sections captured along the Z -axis. These confirmed the

intracellular presence of LCS-NPs (green), which were localized among the actin fibres in the cytoskeleton (red) stained with phalloidin. Scale bar ¼ 25 mm. Images are repre-

sentative of at least three independent experiments. (Taken from Diebold et al., 2007 (Biomaterials), with permission of Elsevier).

Fig. 3. CS NP in vivo uptake. Fluorescence microscopy of ocular surface structures of sham-treated (A, D), CS NP-treated (B, E) and contralateral control (C, F) rabbit eyes.

Representative corneal (A, B, C) and conjunctival (D, E, F) sections are shown. No fluorescence was detected in sham control corneas (A) or conjunctivas (D). (B) Corneal epithelial

cells of the CS NP-treated rabbits were uniformly fluorescent. Inset in (B): zoom area showing details of the corneal epithelium fluorescence pattern. (E) Fluorescence in conjunctival

epithelial cells was intense in the apical cell membranes and positive along the basolateral cell membrane. Inset in (E): zoom area showing the basolateral membrane fluorescence

staining in goblet and non-goblet cells. (C, F) Some fluorescence was detected in corneal and conjunctival epithelial cells from contralateral control eye (OS), although much less

intense than in the treated (OD) eye. Scale bar (AeF)¼ 50 mm; Inset scale bar¼ 10mm. (Taken from Enríquez de Salamanca et al., Invest. Ophthalmol. Vis. Sci., 2006, with permission

of the Association for Research in Vision and Ophthalmology).




results in terms of sustained release and timolol permeation, lacksa complementary toxicity study.

We have recently started working with NPs composed of hya-luronic acid and chitosan (HA-CS NPs) (de la Fuente et al., 2008a).

This new development is capable of encapsulating macromolecules

of both hydrophilic nature, such as the protein bovine serumalbumin, and of hydrophobic nature, such as the polypeptide CsA.Also, HA-CS NPs can carry plasmid DNA and may be suitable for

gene delivery as shown with ocular surface-derived cell lines (de laFuente et al., 2008b). The most important aspect is the good in vivotolerance of these nanosystems (Contreras-Ruiz et al., 2009), whichopens the possibility of testing them in actual disease models.

By exploiting the known properties of HA-CS NPs, our group iscurrently working to design specific nanomedicines that utilizegene therapy to target certain ocular surface inflammatorydiseases.

It is also worth mentioning that primary open-angle glaucoma isthe most widespread neuropathy, affecting 60.5 million people inthe world (Quigley and Broman, 2006). Aside from surgery, the only

partially effective treatment for this disease is IOP-reducing drugsapplied topically onto the cornea. The necessity of numerous dailyapplications of eye-drops for life is a serious issue for patients,besides the risk of side effects in the anterior structures of the eye.

While there was an early interest in drug delivery systems, such asthe OcusertÒ in 1974 for pilocarpine sustained release, there aresurprisingly few reports testing NPs intended for glaucoma treat-ment. Zimmer et al. (1994)tested differentpharmaceuticalaspects of

pilocarpine-loaded PBCA NPs versus a standard pilocarpine solutionin an elevated IOPrabbit model. TheNPs were better in reducing IOPand maintaining miosis, especially at lower drug concentrations,than the drug solution. The NPs induced maximum reduction of IOP

at 2e3 h, whereas the drug solution maximum response was at1e2 h. Epinephrine-loaded poly-N-isopropylacrylamide NPs werealso tested in rabbit to evaluate IOP-lowering effect (Hsiue et al.,2002). The polymer in this nanosystem is thermosensitive and

undergoes a phase transition when the temperature increases toabout 32 C. This allows the progressive release of epinephrine after

being topically administered. The NPs had six times more long-lasting effect compared to conventional eye-drops. Finally,Wadhawa et al. (2009) reported a significant IOP reduction in rabbiteyes exposed to CS-HA NPs loaded with timolol compared to stan-

dard timolol eye-drops or blank NPs. There were no irritant effects.Although not properly NPs but a nanodevice, it is worth mentioninghere thedevelopment of a prototypefor a nanodrainagesystemto beimplanted in the sclera as a bypass route for humour aqueous

outflow (Pan et al., 2006). This new concept might revolutionizeglaucoma treatment in the coming years.

6. NPs and the posterior segment of the eye

Even though the cornea constitutes one of the most selectivebarriers to foreign molecules for the eye, transcorneal penetrationof topically administered ophthalmic medicines intended for the

posterior segment is persistently sought. The reason is thatcurrently, the best way to treat intraocular inflammation, eitherinfectious or non-infectious, is by injecting drugs into the vitreous.

The vitreous is a gelatinous, cell-free structure that is capable of retaining molecules and also delivering them to nearby structures,such as the ciliary body or the RPE, a vital component of the retina.Frequent intraocular injections are needed to treat retinal disor-

ders. With these injections come potential undesired side effects,higher risk of infections, and poor acceptance by the patient. More

frequent side effects associated with repeated intravitreal injec-tions include increased risk of cataract development, vitreous

hemorrhage, retinal detachment, and endophthalmitis.

The prospect of frequent intravitreal injections to treat seriousintraocular disorders affecting the choroid and retina has movedresearchers to look for better solutions derived from the use of NPsas drug carriers. However, the scenario is quite challenging for NPs.

Typically, the cornea is penetrated by less than 5% of drugs applied

as liquid eye-drops (Keister et al., 1991) because of the limitationsmostly imposed by tear turnover associated with blinking and thenasolacrymal drainage system. In addition, the drugs must diffuse

a great distance between the ocular surface and the intraoculartargets. Therefore, usually eye-drops do not provide suf ficient drugconcentration in the posterior ocular tissues. On the other hand,systemic drug administration delivered through the blood vascular

system is not very effective because of the uveal bloodeaqueousand blooderetina barriers. As a consequence, a poor dos-eeresponse profile for vitreoretinal diseases is generally achieved,and a large amount of the drug is needed to maintain therapeutic

levels, usually for insuf ficient amounts of time (Geroski andEdelhauser, 2001). Additionally, the high concentration of drugsneeded to penetrate the bloodeocular barriers is often associated

with systemic side effects.Thus, much effort has been invested in the last decade to opti-mize drug delivery systems for intraocular treatment. Alternativesto intravitreal or periocular injections, including scleral implants

and devices, transdermal patches, and different iontophoreticdevices including hydrogel reservoirs, have been explored withvariable results (for review, see del Amo and Urtti, 2008). However,the ability to achieve long-term drug delivery in the retina and

nearby tissues while reducing the number of intraocular injectionsto just one seems feasible at this time. Several kinds of NPs carryingdiverse active molecules, including genetic material, are currentlyin pre-clinical studies using the above mentioned approaches

(Bourges et al., 2003; Bejjani et al., 2005; Normand et al., 2005;Irache et al., 2005; Farjo et al., 2006; Paasonen et al., 2007 ). Theunderlying idea is to take advantage of the vitreous capacity forretaining and delivering molecules to tissues with which it is in

direct contact and to useit as a biological reservoir once the NPs areplaced inside. If therapeutic levels of a drug can be maintained formonths after a single intravitreal injection, that would be consid-ered an enormous improvement for the quality of life of many

patients.There are promising studies reported in the recent literature on

the use of intravitreally injected NPs. Ganciclovir-loaded albuminNPs are an interesting example. Ganciclovir is one of the standard

treatments for cytomegalovirus retinitis, a prevalent infectiousretinal disease in immunosuppressed patients, such as those withAIDS. In vitro experiments demonstrated that albumin NPs releasedganciclovir in a sustained way (Merodio et al., 2001), witha significant improvement of drug uptake by cytomegalovirus-

infected human cells (Merodio et al., 2002). For single intravitrealinjections in rats, these NPs were safe, well-tolerated carriers notonly for ganciclovir, but also for the anti-cytomegaloviral oligonu-cleotide analog formivirsen. They were present in the vitreous andciliary body for at least two weeks (Irache et al., 2005).

RPE cells have the capacity to take up different kinds of NPs(Bourges et al., 2003; Bejjani et al., 2005; Normand et al., 2005 )opening the possibility of using them to treat retinal disorders

associated with ageing or photoreceptor dystrophies. The purposewill be to target these cells with specific molecules or geneticmaterial capable of reversing or stopping the processes leading tothese diseases. Bourges et al. (2003) tested in rats intravitreal PLA

NPs loaded with fluorochromes and showed a preferential locali-zation at the RPE cells after 24 h. The most interesting achievement

was that RPE cells retained the NPs, which continuously deliveredthe fluorochrome for months after the single injection. Fluores-

cence diffusing from the NPs was observed in distant parts of




retinal tissue, ganglion cells, and rod outer segments for up to fourmonths after the injection. In contrast, detection after injection of the free fluorochrome lasted barely one week. Later, Bejjani et al.(2005) studied in vitro and in vivo PLA and PLGA NPs loaded not

only with fluorochromes but also with model plasmids. NPs

encapsulating a plasmid encoding red nuclear fluorescent proteinwere localized in the RPE cells 24 h after intravitreal injection inrats. Effective plasmid expression was achieved after four days of

injection and expression-associated red fluorescence remaineddetectable in RPE cells during the following three weeks, with noapparent tissue damage or toxicity. In all of these studies, theassociation of the delivered molecules with the NPs and kinetic

release rate profiles were determined prior to the in vivoexperiments.

From a therapeutic point of view, not only is the reduction in thenumber of intravitreal injections by the use of NPs a goal, but the

improvement in intraocular availability of topically applied drugs isan important and remarkably challenging goal. An illustrativeexample is the case of steroidal and non-steroidal anti-inflamma-

tory drugs. There are many clinical situations in which these drugsare normally used. However, because they are almost completelyinsoluble in water and because they are effectively excluded fromintraocular sites by the various bloodeocular barriers, the ability to

reach intraocular targets is quite low. For instance, surgicaltraumas, such as cataract surgery, cause miosis that is treated withnon-steroidal anti-inflammatory drugs. Pignatello et al. (2002)developed ibuprofen-loaded NPs made of inert polymeric resins

(Eudragit RS100) optimized as a pharmaceutical preparation. TheNPs showed a controlled ibuprofen release profile in vitro, and theyhad a high ef ficacy in reducing miosis with an in vivo model of ocular trauma. The therapeutic effect was achieved with lower drug

concentration than in an eye-drop formulation and without anytoxic effect on ocular tissues. Recently, Kassem et al. (2007) studieddifferent nanosuspensions prepared by high-pressure homogeni-zation of three insoluble glucocorticoids: hydrocortisone, prednis-

olone, and dexamethasone. Using normotensive rabbits, theydetermined if the glucocorticoid-associated NPs instilled into thelowercul-de-sac demonstrated enhanced absorption and improvedintensity of drug action. Based on the measured increase in intra-

ocular pressure (IOP), they reported not only improvements inboth, but also a significant extension of the glucocorticoid action.Interestingly, intravenously injected PLA NPs encapsulating beta-methasone phosphate effectively controlled inflammation in a rat

model of experimental autoimmune uveoretinitis (Sakai et al.,2006).

The studies described above have opened a new perspective forthe treatment of retinal and uveal disorders. Nevertheless, to

implement this kind of delivery system in a clinical setting, more

functional studies are needed to exclude any impairment of theretinal function and vision and the development of accompanyingchronic inflammatory processes. Surely, in coming years we shall

see reports dealing with all of these topics.

7. NPs and gene delivery/therapy

The Roadmap for Nanomedicine (http://nihroadmap.nih.gov/nanomedicine/) released by the NIH presents NPs as a strategy toimprove non-viral gene transfer. The eye is an excellent candidate

for gene therapy for two main reasons: it has immune privilege,and it is affected by many well understood genetic-based diseases.

‘Immune privilege’ means that the immune system is driventowards tolerance to foreign antigens rather than rejection and

inflammation, the normal responses. Immune tolerance serves toprotect vision by avoiding the collateral inflammation that is

associated with the immune response against any antigen. Also, it is

a small and closed organ, with very limited diffusion of locallyapplied active molecules to the bloodstream because of thebloodetissue barriers. Hence, there is a growing interest inexploring the suitability of gene delivery strategies in ocular

therapy since the 90s (Nussenblatt and Csaky, 1997; Tanelian et al.,

1997).Genetic-based therapies can be developed using different

nucleic acids such as DNA, antisense oligonucleotides (AS-ODNs),

small interfering RNA(siRNA), and aptamers. AS-ODNs are syntheticmolecules of short sequences, 13e25 nucleotides, that bind tospecific mRNAs. By binding to the mRNA molecules, AS-ODNs arecapable of stopping translation of the mRNA and, consequently,

block the protein synthesis of the targeted gene (Loke et al., 1989).siRNAs share with AS-ODNs the capacity of blocking proteinsynthesis from a given mRNA. However, the gene silencing mech-anism by which it is performed, called RNA interference (RNAi), is

different (Leung and Whittaker, 2005; Bumcrot et al., 2006). RNAi isinduced in mammalian cells by means of synthetic double-strandedsiRNAs. These molecules have small sequences, 21e23 nucleotides,

are highly selective and sequence-specifi

c, and have better stabilitycompared to that of AS-ODNs. Finally, aptamers aresynthetic single-stranded DNA or RNA molecules with a unique 3-D structure. Theyare able to specifically bind other molecules and are particularly

prone to bind the functional domains. This feature makes themuseful as modulators of the targeted molecule (Proske et al., 2005;Nimjee et al., 2005). Therapeutic applications of all of these mole-cules in the eye have been extensively reviewed (Borrás, 2003;

Henry et al., 2004; Campochiaro, 2006; Fattal and Bochot, 2006,2008; Levy-Nissenbaum et al., 2008).

Delivery of genetic material is quite a challenge from a phar-maceutical point of view. It is unstable in biological fluids and has

poor cell penetration due to its size or charge. For instance, plasmidDNA is large; however, siRNA is quite small. These facts imply thatsuitable carriers to deliver it to ocular tissues are needed. A partiallysuccessful approach in recent years has been the useof viral vectors

such as adenovirus, retrovirus, lentivirus, and mainly recombinantadeno-associated virus (rAAV), as gene carriers (Snyder, 1999).rAVV vectors carry single-stranded DNA which is inserted into thegenome of the targeted cell. In general, gene delivery using rAAV

shows a lack of pathogenicity, good long-term transgene expres-sion, and no toxicity. However this technology has limitations suchas lack of effective transduction in some cell types or presence of neutralizing antibodies for some rAVV serotypes (Rabinowitz and

Samulski, 1998; Grimm and Kay, 2003). Also, progress has beenmade to deliver naked DNA to cells (Herweijer and Wolff, 2003).

Different studies have established that some viral vectors canef ficiently deliver transgenes to ocular tissues while others cannot.

For instance, several rAAV serotype vectors appear unsuitable for

anterior segment delivery; however, rAAVs appear to have a selec-tive tropism for retinal ganglion cells (Borrás et al., 2002). There areexamples of effective rescue of genetic deficits in the eye using viral

and rAAV-mediated gene therapy in different in vitro and in vivomodels (Campochiaro, 2002; Martin et al., 2002; Borrás, 2003;Ziche et al., 2004; Ralph et al., 2006; Roy et al., 2010). Oneremarkable use of this technology is the recovery of visual function

in RPE65-deficient dogs. Genetic deficiency of RPE65, a protein

involved in retinoid metabolism in RPE cells, results in blindnesssimilar to human Leber’s Congenital Amaurosis. Recovery of visualfunction in the dogs was achieved after subretinal injection of rAAV encoding RPE65 (Acland et al., 2001). These important results have

led to a gene therapy clinical trial for the RPE65-deficient form of the human disease (Bainbridge et al., 2008; Cideciyan et al., 2008).

However, the use of viral vectors poses risks for the safety of patients. Additionally, the effectiveness in the eye may be limited

due to several factors, including cell tropism, the size of the




sequence to be carried and expressed, and most importantly, hostimmunity (Bennett, 2003). Despite the promising results of virally-mediated ocular gene therapy, non-viral carriers would be thepreferred choice. Still, the issue of an optimal delivery system

remains to be solved. Features that gene delivery systems should

provide for optimal activity of the delivered nucleic acid moleculeinclude improved stability, increased half-life, specific tissue andcellular targeting, improved cellular penetration, and release of the

molecule in the right intracellular compartment (i.e., DNA in thenucleus, however, siRNAs in the cytoplasm).

Hence, gene delivery using non-viral carrier systems holdspromise of being safer and more effective, as those limiting factors

cited above do not influence them so strongly. Several investiga-tions have started using different approaches, including naked DNAdelivery by means of electroporation and the formation of lip-oplexes by DNA condensation with cationic lipids among others

(Kachi et al., 2005; Bloquel et al., 2006; Johnson et al., 2008).Regarding genetic material incorporation to NPs, there are exam-ples using compacted DNA NPs.

Regarding the anterior segment of the eye, our group hasobtained promising results using HA-chitosan oligomers (CSO) NPsloaded with a model plasmid encoding for alkaline phosphatase(manuscript submitted). We are currently studying the intracellular

traf ficking of those nanoparticles and contents in human epithelialcell lines derived from the ocular surface. Our preliminary resultsshow an effective delivery of the plasmid to the cell interior andalkaline phosphatase expression 48 h after transfection using HA-

CSO NPs. Another recent example is the work published byKlausner et al. (2010) in which ultrapure CSO (NOVAFECT) was usedto complex with a model plasmid encoding for green fluorescentprotein (pGFP) to form NPs. Transgene expression of the pGFP was

detected in cell cultures. In rat corneas pGFP was expressed 5.4times that of control polyethylenimine-pGFP NPs. It is worthmentioning that in vivo transgene expression was identified incorneal stroma but not in the epithelium or the endothelium.

Another approach is DNA condensed with polycationic poly-mers. Farjo et al. (2006) packaged compacted DNA in PEG-substituted lysine peptides that formed the NPs. The compactedDNA was a model plasmid encoding for enhanced green fluores-

cent protein (pEGFP), and its expression was studied in mice. Twodays after an intravitreal injection, fluorescence was present in thelens, cornea, trabecular meshwork, sclera, choroid, RPE, and otherretinal cells. However, two days after a subretinal injection, fluo-

rescence was restricted to retina and RPE cells, choroid, and sclera,with a minimal presence in the lens. The authors reported noalteration in visual function, evaluated by electroretinography, andno evidence of inflammation in the histological analysis of the

ocular tissues. In this study, the duration of DNA expression was

not reported. Ding et al. (2009) recently reported the preparationof single molecule of pEGFP compacted with PEG-substitutedpolysysine (CK30PEG). The formed NPs were subretinally injected

in mice and resulted in ef ficient retinal cells transfection. Thiswork was more focused on toxicity issues related with that tech-nology. There were no signs of local inflammatory response interms of infiltration of inflammatory cells or chemokine marker

expression. Even though this method of gene therapy is poten-tially applicable for multiple ocular diseases, a more directed tar-geting is desirable.

A quite recent concept is the use of light-sensitive NPs made bycombining a structural protein of the Herpes simplex virus, VP22,with AS-ODNs bound through the C-terminal end of the viralprotein. This leads to the formation of spherical NPs of 0.3e1 mm in

diameter named ‘vectosomes’. AS-ODNs selectively modulate theexpression of a given gene by displaying a base sequence that is

complementary to a specific mRNA (Helene and Toulme, 1990). The

precise local delivery of bound ODNs in target cells is controlled byilluminating them (Normand et al., 2001; Zavaglia et al., 2003). Thistechnology, used for tumour cells, is particularly interesting inophthalmology due to the frequent use of lasers for therapeutic

purposes. Light-induced delivery of AS-ODNs from vectosomes has

been studied in vitro, using human melanoma and RPE cell lines,and in vivo with rats (Normand et al., 2005). Interestingly, thevectosomes followed a rapid transretinal migration pattern after

intravitreal injection in rats, and they were internalized by RPE cellsand other cell types. Transscleral illumination of injected eyesinduced disruption of the light-sensitive vectosomes and migrationof released AS-ODNs to the cell nucleus, where they were localized

in a light-dependent manner. Not only were the AS-ODNs localizedin the RPE cells, they were also detected in ganglion cells, innernuclear layer, and even in the choroid. Much work is needed tolearn about the light wavelengths and energy produced as

a consequence of vectosome rupture, which can potentially harmthe retina. However, this approach offers great therapeuticpotential.

Afi

nal example of potential gene therapy treatment of retinaldiseases is the recent work of Park et al. (2009). They encapsulatedin PLGA NPs an expression plasmid for a natural angiogenicinhibitor, plasminogen kringle 5 (K5) that inhibits ischemia-

induced neovascularisation in a rat model of oxygen-induced reti-nopathy (OIR). After administering K5-loaded NPs intravitreally toanimals with OIR, the authors reported high K5 expression in theinner retina for four weeks. Also, retinal vascular leakage and retina

neovascularisation were reduced in those K5-NP injected eyeswhen compared to fellow control eyes.

Even though these possibilities make those of us who work inthe development of new therapies in ophthalmology dream of the

end of many handicapping disorders, many challenges remain. Forinstance, we must consider the potential capacity of the vitreous toact as a barrier for gene delivery, mainly due to its composition andbiological characteristics that affect diffusion of large molecules

(Xu et al., 2000; Peeters et al., 2005). We need a better under-standing of the biological processes affecting intraocular structuresto help design more specific solutions. The identification of moretarget genes involved in the development of each pathological

condition is necessary. We must learn about potential immuneresponses derived from the use of different non-viral vectors. Forpre-clinical studies, it is important to have available and selectcarefully appropriate animal models of the target disease. Finally,

we will need to identify those elements that control the long-termexpression of the delivered transgenes or the permanent shuttingoff of genes with delivered AS-ODNs.

8. Nanoparticle safety: toxicity and interaction with the

immune system

Not just ocular, but any biomedical application of NPs as

a therapeutic agent requires biocompatibility. This means that NPs,both the components and the assembled NP itself, need to bebiologically compatible with living tissues by not producing toxic,

injurious, or immunological responses in them. Key aspects influ-encing the biocompatibility of NPs are the physicochemical char-acteristics, such as size, shape, charge, solubility, and chemicalgroups on the surface that provide particle charge and lipo- or

hydro-phobic features (McNeil, 2005). However, the same proper-ties that make NPs attractive for biomedical applications may makethem reactive in biological systems and develop toxicity. Forinstance, smaller size NPs are preferred for better interactions at

the cellular level. However, smaller NPs have larger surface area perunit mass, which may mean higher reactivity and consequently, cellor tissue toxicity (Kipen and Laskin, 2005).




Risks posed by both organic and inorganic NPs include aggre-gation,tissue accumulation, and adsorption of plasma proteins ontothe surface. This latterconsideration is particularly important whenthinking about an intravenous NP administration or a potential

access of theblood systemusing other non-systemic administration

routes, e.g., intraocular administration to treat posterior segmentdiseases that involve blooderetina barrier impairment. NP aggre-gation mayblockcell metabolismor even impairtissue function. For

instance, aggregation of topically applied NPs onto the ocularsurface may block the lachrymal drainage punctum and impair tear

film recycling. Additionally, indiscriminate NP accumulation inocular tissues may distort tissue architecture and consequently,

alter function. Finally, there may be toxic effects due to the presenceof high levels of the loaded drug in a non-target tissue.

Potential cytotoxic activity of NPs may include alterations of cellmembranes such as membrane disruption, as has been described

for carbon nanotubes (Panessa-Warren et al., 2009). However,sometimes this particular property may be sought, especially forgene delivery (Kiang et al., 2004; Akagi et al., 2010). For instance,

Kiang et al. (2004) used poly (propyl acrylic acid) to formulatechitosan-DNA NPs with enhanced in vitro transfection ef ficiency.That polymer was specifically designed to disrupt the lipid bilayerin cell membranes. By changes in the pH, it triggered the release of

DNA from the endosomal compartment. Very recently, Akagi et al.(2010) evaluated the relationship of different physicochemicalcharacteristics of 200 nm-size NPs composed of poly(gamma-glu-tamic acid). The protein-loaded NPs had significant haemolytic

activity in erythrocytes, depending on NP hydrophobicity and pH,with the greatest activity present at pH 7 to 5.5 and absent atphysiological pH.

Noxious effects of different kinds of NPs have been reported in

several organ systems (for review please see Medina et al., 2007).One of the main mechanisms described by which NPs may harmcells and tissues is oxidative stress generation, which in turn, maylead to the activation of different transcription factors (Medina

et al., 2007). Generally, NPs can be taken up by lymphatic nodesand distributed through the lymphatic system in parallel with theblood vascular system. The ocular mucosa possesses lymphoid

tissue that drains to different face and neck lymphatic ganglia. TWProw (2009) recently published a very comprehensive review

about the toxicity of nanomaterials in the eye. He nicely summa-rized in vitro and in vivo relevant studies accomplished since 1996for about thirty different kinds of nanoparticles and other nano-

carriers testedfor ocular applications. The types of toxicity reportedincluded cell morphology and viability, clinical signs evaluation,gross tissue examination, irritation test, histology and functionalanalyses, and inflammatory response (Table 4). That review high-lights the importance of accomplishing toxicity testing of newly

developed drug carrier candidates.Another consideration is the potential inflammatory, immu-

nostimulatory, and immunosuppressive properties described fordifferent kinds of NPs (Dobrovolskaia and McNeil, 2007; Zolnik

et al., 2010). There are limited data available about this topic(Zolnik et al., 2010), but it is known that some NPs are antigenicthemselves. The antigenicity depends on particle size, especiallythose of ultra-small size (25 nm or smaller) that improves

lymphatic uptake, and surface charge (Reddy et al., 2007; Manolova

et al., 2008). Allergic or hypersensitivity reactions can be induced oraggravated in animal models and humans by dendrimers (Toyama

et al., 2008), carbon nanotubes (Nygaard et al., 2009), lipid-basedNPs (Szebeni et al., 2007), titanium dioxide NPs (Yanagisawa et al.,2009), and polystyrene NPs (Yanagisawa et al., 2010). However, it isimportant to bear in mind that sometimes NPs are specificallydesigned to target the immune system, and interactions withimmune cells are considered beneficial. Such is the case for those

NPs intended for vaccine development, in which the immunogenicproperties are exploited. NPs can serve as adjuvants as they can beconjugated with antigens.

Currently, a major issue related to the development of NP-based

novel therapies is the rigorous evaluation of the potential immu-notoxic effects. Researchers in the field of nanomedicine agree thatthe potential environmental and health-related risks should becarefully analyzed. Importantly, we, in the eye community, test NPs

for toxic effects less than we should. The reasons for that are many,but the most important one is the absence of a common regulatoryframework. Technological advancements develop faster than

Table 4

Summary of key questions to be addressed during early phase pre-clinical evaluation of nanoparticles (NPs) intended for use in biomedical applications.

Assay category Questions to address

In vitro

Hemolysis Do NPs change integrity of red blood cells?

Platelet aggregation Do NPs interfere with cellular components of the blood coagulation cascade?

Coagulation time Do NPs cause changes in the function of the coagulation factors?

Complement activation Do NPs activate the complement system?

Colony-forming unit granulocyte macrophage Do NPs cause myelosuppression (toxicity to bone marrow precursors)?Leukocyte proliferation Do NPs have adverse effects on leukocyte proliferative responses?

Uptake by macrophages Are NPs internalized by specialized phagocytes?

Cytokine induction Do NPs activate immune cells to elicit cytokine production or interfere with that

caused by known immunogens?

Nitr ic oxide pr oduction Do NPs induce oxidative str ess? Indirect test for potential endotoxin contaminat ion

Cytotoxicity of natural killer cells Do NPs interfere with the ability of natural killer cells to recognize and kill tumour target cells?

Endotoxin contamination Pyrogen contamination test

Microbial/viral/ myco plasma cont amination Ster il ity te st

In vivo

Single-dose toxicity study: These tests aim at answering the following questions:

Standard toxicity tests (blood chemistry, hematology,

histopathology, gross pathology)

Do NPs cause toxicity to immune cells and organs? Are there any indications

for additional toxicity studies?

Additional studies are conducted on a case-by-case basis using weight-of-evidence approach

T cell dependent antibody response (TDAR) This test is recognized for its high predictability for human models

Host resistance studies; evaluation of cell-mediated immunity These tests are recommended for 1) testing the potential effects that particles might have on

host resistance towards pathogens and tumour cells, and 2) to check for contact sensitization

and delayed type hypersensitivity reactionsRepeated dose toxicity study; immunogenicity Do NPs elicit particle specific immune response?

According to Dobrovolskaia & McNeil (Nature Nanotech., 2007).




regulations in the nanomedicine field. Standardization of proce-dures to study immunological properties and toxicology-relatedissues would also help. There certainly are regulations in Europe,U.S., and Japan intended to assess the immunotoxic potential of

newly developed pharmaceuticals (Putman et al., 2003; Snodin,

2004). However, there are no specific protocols for those nano-technology-based tests because the properties of the NPs mayinterfere with the established tests (Stone et al., 2009). A first

step towards that purpose has been taken by the NationalCharacterization Laboratory, U.S. National Cancer Institute (http://ncl.cancer.gov/working_assay-cascade.asp), whose mission is toperform and standardize the pre-clinical characterization of

nanomaterials intended for cancer therapeutics and diagnostics. Asan example, in a recent review Dobrovolskaia and McNeil (2007)suggested the most important parameters that need to beaddressed during an initial evaluation of new nanotechnology-

derived pharmaceuticals (Table 4).

9. Future directions

A novel direction for nanomedical applications involves nano-ceria particles. This unique type of NP is made of nanocrystallinecerium oxide, CeO2, also known as ceria. It is a rare earth oxide from

the lanthanide series of the periodic table. Properly speaking,nanoceriaparticlesdo notdeliverany drug;ratherthey arethe drugs.These NPs can scavenge free radicals and reactive oxygen species(Heckert et al., 2008). Interest in them for biomedical applications

derives from several in vitro studies in which they increased thelifespan of cultured brain cells (Rzigalinski et al., 2003), protectednon-tumoral cells from radiation therapy effects (Tarnuzzer et al.,2005), and protected in vitro rat spinal cord neurons from oxida-

tive stress (Das et al., 2007). For ocular applications, there are onlya few studies with promising results. One of them, by Chen et al.(2006), showed that nanoceria particles were effective in the inhi-bition of the reactive oxygen intermediate-induced photoreceptor

cell death. As macular degenerationand retinitis pigmentosa,amongother blinding diseases, are thought to generate reactive oxygenspecies, theuse of nanoceria may be a useful therapeutic strategy. Inanother study, Pierscionek et al. (2010) showed the potential of

antioxidant nanoceria particles in cataract treatments. It is certainthat more studies will arise in thenearfuture to explorethe potentialbenefit of nanoceria particles in different eye diseases.

Other inorganic NPs made of noble metals,such as gold (Hayashi

et al., 2009) orsilver (Gurunathan et al., 2009), aregenerating muchinterest dueto their small size, about20 nm, and the great potentialof traversing the blooderetina barrier (Kim et al., 2009). There arevery few reports, and most of them show preliminary results. More

and deeper studies using these kinds of small NPs are necessary to

understand the pharmacokinetics and clearance mechanisms(Amrite et al., 2008) and the actual therapeutic potential.

Also, the use of inorganic NPs as novel contrast agents for

molecular imaging in other tissues suggests that this technologycan be applied to ocular imaging. A few interesting examples of NPsused as imaging agents are gadolinium-loaded nanoemulsions forbrain imaging, gold NPs for optical coherence tomography imaging,

and superparamagnetic iron oxide NPs with gold shells formagnetic resonance imaging. Regarding ocular tissues, Yamamotoet al. (2007) have shown that quantum dots can be used forimagingthe vitreous. No doubt, the coming years will bring exciting

developments in this field.

10. Summary and conclusions

It is currently possible to design nanocarriers with specificdelivery requirements for ocular administration. Those carriers are

able to safely deliver the loaded therapeutic molecule while pre-venting damage or deactivation of it. The loaded agents can act

more ef ficiently and with fewer side effects when compared to thesame agents administered without the nanocarrier. However, manyquestions still remain. It is an urgent matter to resolve them so that

new and more ef ficient drug formulations based on NP technologyfor ocular therapy can be made available for patient care.

Author disclosure statement

The authors report nofinancial interest in any of the materials ornanosystems presented in this review.

Acknowledgements

This work was supported by a Spanish Ministry of Science and

Technology Grant (MAT2007-64626-C02) and the NetworkingResearch Centre on Bioengineering, Biomaterials and Nano-medicine (CIBER-BBN), Spain. CIBER-BBN is an initiative funded by

the VI National R&D&I Plan 2008e

2011, Iniciativa Ingenio 2010,Consolider Program, CIBER Actions and financed by the Instituto de

Salud Carlos III with assistance from the European Regional Devel-opment Fund.

References

Acland, G.M., Aguirre, G.D., Ray, J., Zhang, Q., Aleman, T.S., Cideciyan, A.V., Pearce-Kelling, S.E., Anand, V., Zeng, Y., Maguire, A.M., Jacobson, S.G., Hauswirth, W.W.,Bennet, J., 2001. Gene therapy restores vision in a canine model of childhoodblindness. Nat. Genet. 28, 92e95.

Aggarwal, P., Hall, J.B., McLeland, C.B., Dobrovolskaia, M.A., McNeil, S.E., 2009.Nanoparticle interaction with plasma proteins as it relates to particle bio-distribution, biocompatibility and therapeutic ef ficacy. Adv. Drug Deliv. Rev. 61,428e437.

Akagi, T., Kim, H., Akashi, M., 2010. pH-dependent disruption of erythrocyte

membrane by amphiphilic poly(amino acid) nanoparticles. J. Biomater. Sci.Polym. Ed. 21, 315e328.

Alonso, M.J., Sánchez, A., 2003. The potential of chitosan in ocular drug delivery. J. Pharm. Pharmacol. 55, 1451e1463.

Amrite, A.C., Edelhauser, H.F., Singh, S.R., Kompella, U.B., 2008. Effect of circulationon the disposition and ocular tissue distribution of 20 nm nanoparticles afterperiocular administration. Mol. Vis. 14, 150e160.

Attama, A.A., Reichl, S., Müller-Goymann, C.C., 2009. Sustained release andpermeation of timolol from surface-modified solid lipid nanoparticles throughbioengineered human cornea. Curr. Eye Res. 34, 698e705.

Bareford, L.M., Swaan, P.W., 2007. Endocytic mechanisms for targeted drug delivery.Adv. Drug Deliv. Rev. 59, 748e758.

Bayens, R., Gurny, R., 1997. Chemical and physical parameters of tears relevant forthe design of ocular drug delivery formulations. Pharm. Acta Helv. 72,191e202.

Bejjani, R.A., BenEzra, D., Cohen, H., Rieger, J., Andrieu, C., Jeaanny, J.-C., Golomb, G.,Behar-Cohen, F.F., 2005. Nanoparticles for gene delivery to retinal pigmentepithelial cells. Mol. Vis. 11, 124e132.

Bennett, J., 2003. Immune following intraocular delivery of recombinant viralvectors. Gene Ther. 10, 977e982.

Bloquel, C., Bourges, J.L., Touchard, E., Berdugo, M., BenEzra, D., Behar-Cohen, F.F.,2006. Non-viral ocular gene therapy: potential ocular therapeutic avenues. Adv.Drug Deliv. Revs. 58, 1224e1242.

Borrás, T., 2003. Recent developments in ocular gene therapy. Exp. Eye Res. 76,643e652.

Borrás, T., Brandt, C.R., Nickells, R., Ritch, R., 2002. Gene therapy for glaucoma:treating a multifaceted, chronic disease. Invest. Ophthalmol. Vis. Sci. 43,2513e2518.

Bourges, J.L., Gautier, S.E., Delie, F., Bejjani, R.A., Jeanny, J.-C.-, Gurny, R., BenEzra, D.,Behar-Cohen, F.F., 2003. Ocular drug delivery targeting the retina and retinalpigment epithelium using polylactide nanoparticles. Invest. Ophthalmol. Vis.Sci. 44, 3562e3569.

Bainbridge, J.W., Smith, A.J., Barker, S.S., Robbie, S., Henderson, R., Balaggan, K.,Viswanathan, A., Holder, G.E., Stockman, A., Tyler, N., Petersen-Jones, S.,Bhattacharya, S.S., Thrasher, A.J., Fitzke, F.W., Carter, B.J., Rubin, G.S., Moore, A.T.,Ali, R.R., 2008. Effect of gene therapy on visual function in Leber ’s congenitalamaurosis. N. Engl. J. Med. 358, 2231e2239.

Bumcrot, D., Manoharan, M., Koteliansky, V., Sah, D.W., 2006. RNAi therapeutics:

a potential new class of pharmaceutical drugs. Nat. Chem. Biol. 2, 711e

719.Calvo, P., Sánchez, A., Martínez, J., López, M.I., Calonge, M., Pastor, J.C., Alonso, M.J.,1996. Polyester nanocapsules as new topical ocular delivery systems forcyclosporin A. Pharm. Res. 13, 311e315.




Campochiaro, P.A., 2002. Gene therapy for retinal and choroidal diseases. ExpertOpin. Biol. Ther. 2, 537e544.

Campochiaro, P.A., 2006. Potential applications for RNAi to probe pathogenesis anddevelop new treatments for ocular disorders. Gene Ther. 13, 559e562.

Cideciyan, A.V., Aleman, T.S., Boye, S.L., Schwartz, S.B., Kaushal, S., Roman, A.J.,Pang, J.J., Sumaroka, A., Windsor, E.A., Wilson, J.M., Flotte, T.R., Fishman, G.A.,Heon, E., Stone, E.M., Byrne, B.J., Jacobson, S.G., Hauswirth, W.W., 2008.

Human gene therapy for RPE65 isomerase deficiency activates the retinoidcycle of vision but with slow rod kinetics. Proc. Natl. Acad. Sci. U.S.A. 105,15112e15117.

Chen, J., Patil, S., Seal, S., McGinnis, J.F., 2006. Rare earth nanoparticles preventretinal degeneration induced by intracellular peroxides. Nat. Nanotechnol. 1,142e150.

Conti, S., Polonelli, L., Frazzi, R., Artusi, M., Bettini, R., Cocconi, D., Colombo, P., 2000.Controlled delivery of biotechnological products. Curr. Pharm. Biotechnol. 1,313e323.

Contreras-Ruiz, L., de la Fuente, M., García-Vázquez, C., Sáez, V., Seijo, B.,Alonso, M.J., Calonge, M., Diebold, Y., 2010. Ocular tolerance to a topicalformulation of hyaluronic acid and chitosan-based nanoparticles. Cornea 29,550e558.

Couvreur, P., Tulkenst, P., Roland, M., Trouet, A., Speiser, P., 1977. Nanocapsules:a new type of lysomotropic carrier. FEBS Lett. 84, 323e326.

Das, M., Patil, S., Bhargava, N., Kang, J.-F., Riedel, L.M., Seal, S., et al., 2007. Auto-catalytic ceria nanoparticles offer neuroprotection to adult rat spinal cordneurons. Biomaterials 28, 1918e1925.

de Campos, A.M., Diebold, Y., Carvalho, E.L.S., Sánchez, A., Alonso, M.J., 2004. Chi-tosan nanoparticles as new ocular drug delivery systems: in vitro stability,in vivo fate, and cellular toxicity. Pharm. Res. 21, 803e810.

de Campos, A.M., Sánchez, A., Alonso, M.J., 2001. Chitosan nanoparticles: a newvehicle for the improvement of the delivery of drugs to the ocular surface.Application to cyclosporine A. Int. J. Pharm. 224, 159e168.

de Campos, A.M., Sánchez, A., Gref, R., Calvo, P., Alonso, M.J., 2003. The effect of a PEG versus a chitosan coating on the interaction of drug colloidal carriers withthe ocular mucosa. Eur. J. Pharm. Sci. 20, 73e81.

de la Fuente, M., Seijo, B., Alonso, M.J., 2008a. Novel hyaluronan-based nanocarriersfor transmucosal delivery of macromolecules. Macromol. Biosci. 8, 441e450.

de la Fuente, M., Seijo, B., Alonso, M.J., 2008b. Novel hyaluronic acid-chitosannanoparticles for ocular gene delivery. Invest. Ophthalmol. Vis. Sci. 49,2016e2024.

del Amo, E.M., Urtti, A., 2008. Current and f uture ophthalmic drug delivery systems.A shift to the posterior segment. Drug Discov. Today 13, 135e143.

del Pozo-Rodríguez, A., Delgado, D., Solinís, M.A., Gascón, A.R., Pedraz, J.L., 2008.Solid lipid nanoparticles for retinal gene therapy: transfection and intracellulartraf ficking in RPE cells. Int. J. Pharm. 360, 177e183.

Diebold, Y., Jarrín, M., Sáez, V., Carvalho, E.L.S., Orea, M., Calonge, M., Seijo, B.,Alonso, M.J., 2007. Ocular drug delivery by liposomeechitosan nanoparticlecomplexes (LCS-NP). Biomaterials 28, 1553e1564.

Ding, X.Q., Quiambao, A.B., Fitzgerald, J.B., Cooper, M.J., Conley, S.M., Naash, M.I.,2009. Ocular delivery of compacted DNA-nanoparticles does not elicit toxicityin the mouse retina. PLoS ONE 4, 1e11.

Dobrovolskaia, M.A., Aggarwal, P., Hall, J.B., McNeil, S.E., 2008. Preclinical studies tounderstand nanoparticles interaction with the immune system and its potentialeffects on nanoparticles biodistribution. Mol. Pharmacol. 5, 487e495.

Dobrovolskaia, M.A., McNeil, S.E., 2007. Immunological properties of engineerednanomaterials. Nat. Nanotechnol. 2, 469e478.

Degim, I.T., Celebi, N., 2007. Controlled delivery of peptides and proteins.Curr. Pharm. Des. 13, 99e117.

Edwards, A., Prausnitz, M.R., 2001. Predicted permeability of the corneal to topicaldrugs. Pharm. Res. 18, 1497e1508.

Enríquez-de-Salamanca, A., Diebold, Y., Calonge, M., García-Vázquez, C., Callejo, S.,Vila, A., Alonso, M.J., 2006. Chitosan nanoparticles as a potential drug deliverysystem for the ocular surface: toxicity, uptake mechanism and in vivo tolerance.Invest. Ophthalmol. Vis. Sci. 47, 1416e1425.

European Science Foundation Forward Look on Nanomedicine. http://www.esf.org/publications/forward-looks.html .

Farjo, R., Skaggs, J., Quiambao, A.B., Cooper, M.J., Naash, M.I., 2006. Ef ficient non-viral ocular gene transfer with compacted DNA nanoparticles. PLoS ONE 1 e38.

Fattal, E., Bochot, A., 2006. Ocular delivery of nucleic acids: antisense oligonucle-otides, aptamers and siRNA. Adv. Drug Dev. Rev. 58, 1203e1223.

Fattal, E., Bochot, A., 2008. State of the art and perspectives for the delivery of antisense oligonucleotides and siRNA by polymeric nanocarriers. Intl. J. Pharm.364, 237e248.

Frenkel, V., 2008. Ultrasound mediated delivery of drugs and genes to solid tumors.Adv. Drug Deliv. Rev. 30, 1193e1208.

Gaudana, R., Jwala, J., Boddu, S.H.S., Mitra, A.K., 2009. Recent perspectives in oculardrug delivery. Pharm. Res. 26, 1197e1216.

Geroski, D.H., Edelhauser, H.F., 2001. Transscleral drug delivery for posteriorsegment diseases. Adv. Drug Deliv. Revs. 52, 37e48.

Gref, R., Minamitake, Y., Peracchia, M.T., Trubetskoy, V., Torchilin, V., Langer, R., 1994.Biodegradable long-circulating polymeric nanospheres. Science 263,1600e1603.

Grimm, D., Kay, M.A., 2003. From virus evolution to vector revolution: use of naturally occurring serotypes of adeno-associated virus (AAV) as novel vectorsfor human gene therapy. Curr. Gene Ther. 3, 281e304.

Gurunathan, S., Lee, K.J., Kalishwaralal, K., Sheikpranbabu, S., Vaidyanathan, R.,Eom, S.H., 2009. Antiangiogenic properties of silver nanoparticles. Biomaterials30, 6341e6350.

Heckert, E.G., Karakoti, A.S., Seal, S., Self, W.T., 2008. The role of cerium redox statein the SOD mimetic activity of nanoceria. Biomaterials 29, 2705e2709.

Hämäläinen, K.M., Kananen, K., Auriola, S., Kontturi, K., Urtti, A., 1997. Character-ization of paracellular and aqueous penetration routes in cornea, conjunctiva,

and sclera. Invest. Ophthalmol. Vis. Sci. 38, 627e

634.Harush-Frenkel, O., Altschuler, Y., Benita, S., 2008. Nanoparticle-cell interactions:

drug delivery implications. Crit. Rev. Ther. Drug Carrier Syst. 25, 485e544.Hayashi, A., Naseri, A., Pennesi, M.E., de Juan Jr., E., 2009. Subretinal delivery of

immunoglobulin G with gold nanoparticles in the rabbit eye. Jpn. J. Ophthalmol.53, 249e256.

Helene, C., Toulme, J.J., 1990. Specific regulation of gene expression of by anti-sense, sense and antigenic nucleic acids. Biochim. Biophys. Acta 1049,99e125.

Henry, S.P., Marcusson, E.G., Vincent, T.M., Dean, N.M., 2004. Setting sights on thetreatment of ocular angiogenesis using antisense oligonucleotides. TRENDSPharm. Sci. 25, 523e527.

Herweijer, H., Wolff, J.A., 2003. Progress and prospects: naked DNA gene transferand therapy. Gene Ther. 10, 453e458.

Hillaireau, H., Couvreur, C., 2009. Nanocarriers’ entry into the cell: relevance to drugdelivery. Cell. Mol. Life Sci. 66, 2873e2896.

Hsiue, G.H., Hsu, S.H., Yang, C.C., Lee, S.H., Yang, I.K., 2002. Preparation of controlledrelease ophthalmic drops, for glaucoma therapy using thermosensitive poly-N-isopropylacrylamide. Biomaterials 23, 457e462.

Irache, J.M., Merodio, M., Arnedo, A., Campanero, M.A., Mirshahi, M., Espuelas, S.,2005. Albumin nanoparticles for the intravitreal delivery of anticytomegaloviraldrugs. Mini Rev. Med. Chem. 5, 293e305.

Jarvinen, K., Jarvinen, T., Urtti, A., 1995. Ocular absorption following topical delivery.Adv. Drug Deliv. Rev. 16, 3e19.

Johnson, L.N., Cashman, S.M., Kumar-Singh, R., 2008. Cell-penetrating peptide forenhanced delivery of nucleic acids and drugs to ocular tissues including retinaand cornea. Mol. Ther. 16, 107e114.

Juberías, J.R., Calonge, M., Gómez, S., López, M.I., Calvo, P., Herreras, J.M.,Alonso, M.J., 1998. Ef ficacy of topical cyclosporine-loaded nanocapsules onkeratoplasty rejection model in the rat. Curr. Eye Res. 17, 39e46.

Kachi, S., Oshima, Y., Esumi, N., Kachi, M., Rogers, B., Zack, D.J., Campochiaro, P.A.,2005. Nonviral ocular gene transfer. Gene Ther. 12, 843e851.

Kao, H.-J., Lin, H.-R., Lo, Y.-L., Yu, S.-P., 2006. Characterization of pilocarpine-loadedchitosan/carbopol nanoparticles. J. Pharmacokinet. Pharmacodyn. 58, 179e186.

Kassem, M.A., AbdelRahman, A.A., Ghorab, M.M., Ahmed, M.B., Khalil, R.M., 2007.Nanosuspensions as an ophthalmic delivery system for certain glucocorticoiddrugs. Int. J. Pharm. 340, 126e133.

Kaur, I.P., Kanwar, M., 2002. Ocular preparations: the formulation approach. DrugDev. Ind. Pharm. 28, 473e493.Keister, J.C., Cooper, E.R., Missel, P.J., Lang, J.C., Hager, D.F., 1991. Limits on optimizing

ocular drug delivery. J. Pharm. Sci. 80, 50e53.Kiang, T., Brigth, C., Cheung, C.Y., Stayton, P.S., Hoffman, A.S., Leong, K.W., 2004.

Formulation of chitosan-DNA nanoparticles with poly(propyl acrylic acid)enhances gene expression. J. Biomater. Sci. Polym. Ed. 15, 1405e1421.

Kim, J.H., Kim, J.H., Kim, K.W., Kim, M.H., Yu, Y.S., 2009. Intravenously administeredgold nanoparticles pass through the blooderetinal barrier depending on theparticle size, and induce no retinal toxicity. Nanotechnology 20, 505101.

Kipen, H.M., Laskin, D.L., 2005. Smaller is not always better: nanotechnology yieldsnanotoxicology. Am. J. Physiol. Lung Cell Mol. Physiol. 289, L696eL697.

Klausner, E.A., Zhang, Z., Chapman, R.L., Multack, R.F., Volin, M.V., 2010. Ultrapurechitosan oligomers as carriers for corneal gene transfer. Biomaterials 31,1814e1820.

Leung, R.K.M., Whittaker, P.A., 2005. RNA interference: from gene silencing to gene-specific therapeutics. Pharmacol. Ther. 107, 222e239.

Levy-Nissenbaum, E., Radovic-Moreno, A.F., Wang, A.Z., Langer, R., Farokhzad, O.C.,2008. Nanotechnology and aptamers: applications in drug delivery. Trends

Biotechnol. 26, 442e

449.Li, V.H., Wood, R.W., Kreuter, J., Harmia, T., Robinson, J.R., 1986. Ocular drug delivery

of progesterone using nanoparticles. J. Microencapsul. 3, 213e218.Loke, S.L., Stein, C.A., Zhang, X.H., Mori, K., Nakanishi, M., Subasinghe, C., Cohen, J.S.,

Neckers, L.M., 1989. Characterization of oligonucleotide transport into livingcells. Proc. Natl. Acad. Sci. U.S.A. 86, 3474e3478.

Losa, C., Marchal-Heussler, L., Orallo, F., Vila-Jato, J.L., Alonso, M.J., 1993. Design of new formulations for topical ocular administration: polymeric nanocapsulescontaining metipranolol. Pharm. Res. 10, 80e87.

Losa, C., Calvo, P., Castro, E., Vila-Jato, J.L., Alonso, M.J., 1991. Improvement of ocularpenetration of amikacin sulphate by association to poly(butylcyanoacrylate)nanoparticles. J. Pharm. Pharmacol. 43, 548e552.

Manolova, V., Flace, A., Bauer, M., Schwarz, K., Saudan, P., Bachmann, M.F., 2008.Nanoparticles target distinct dendritic cell populations according to their size.Eur. J. Immunol. 38, 1404e1413.

Mannermaa, E., Vellonen, K.S., Urtti, A., 2006. Drug transport in corneal epitheliumand blooderetina barrier: emerging role of transporters in ocular pharmaco-kinetics. Adv. Drug Deliv. Rev. 58, 1136e1163.

Martin, K.R., Klein, R.L., Quigley, H.A., 2002. Gene delivery to the eye using adeno-associated viral vectors. Methods 28, 267e275.

McNeil, S.E., 2005. Nanotechnology for the biologist. J. Leukoc. Biol. 78, 585e594.




Medina, C., Santos-Martínez, M.J., Radomski, A., Corrigan, O.I., Radomski, M.W.,2007. Nanoparticles: pharmacological and toxicological significance. Br. J.Pharmacol. 150, 552e558.

Merodio, M., Arnedo, A., Renedo, M.J., Irache, J.M., 2001. Ganciclovir-loaded nano-particles: characterization and in vitro release properties. Eur. J. Pharm. Sci. 12,251e259.

Merodio, M., Espuelas, M.S., Mirshahi, M., Arnedo, A., Irache, J.M., 2002. Ef ficacy of

ganciclovir-loaded nanoparticles in human cytomegalovirus (HCMV)-infectedcells. J. Drug Target. 10, 231e238.

Mo, Y., Barnett, M.E., Takemoto, D., Davidson, H., Kompella, U.B., 2007. Humanserum albumin nanoparticles for ef ficient delivery of Cu, Zn superoxide dis-mutase gene. Mol. Vis. 23, 746e757.

Motwani, S.K., Chopra, S., Talegaonkar, S., Kohli, K., Ahmad, F.J., Khar, R.K., 2008.Chitosan-sodium alginate nanoparticles as submicroscopic reservoirs for oculardelivery: formulation, optimisation and in vitro characterisation. Eur. J. Pharm.Biopharm. 68, 513e525.

National Characterization Laboratory, U.S. National Cancer Institute. http://ncl.cancer.gov/working_assay-cascade.asp , (accessed June 2010).

National Institutes of Health of the U.S.A., Nanomedicine Roadmap Initiative.http://nihroadmap.nih.gov/nanomedicine /.

Nimjee, S.M., Rusconi, C.P., Sullenger, B.A., 2005. Aptamers: an emerging class of therapeutics. Annu. Rev. Med. 56, 555e583.

Normand, N., Valamanesh, F., Savoldelli, M., Mascarelli, F., BenEzra, D., Courtois, Y.,Behar-Cohen, F.F., 2005. VP22 light controlled delivery of oligonucleotides toocular cells in vitro and in vivo. Mol. Vis. 11, 184e191.

Normand, N., van Leeuwen, H., O’Hare, P., 2001. Particle formation by a conserveddomain of the Herpes simplex virus protein VP22 facilitating protein andnucleic acid delivery. J. Biol. Chem. 276, 15042e15050.

Nussenblatt, R.B., Csaky, K., 1997. Perspectives on gene therapy in the treatment of ocular inflammation. Eye (Lond) 11, 217e221.

Nygaard, U.C., Hansen, J.S., Samuelsen, M., Alberg, T., Marioara, C.D., Lovik, M., 2009.Single-walled and multi-walled carbon nanotubes promote allergic immuneresponses in mice. Toxicol. Sci. 109, 113e123.

Paasonen, L., Laaksonen, T., Johans, C., Yliperttula, M., Kontturi, K., Urtti, A., 2007.Gold nanoparticles enable selective light-induced contents release from lipo-somes. J. Control Release 11, 86e93.

Pan, T., Brown, D.J., Ziaie, B., 2006. An artificial nano-drainage implant (ANDI) forglaucoma treatment. In: Conf. Proc. IEEE Eng. Med. Biol. Soc., vol. 1, pp.3174e3177.

Panessa-Warren, B.J., Maye, M.M., Warren, J.B., Crosson, K.M., 2009. Single walledcarbon nanotube reactivity and cytotoxicity following extended aqueousexposure. Environ. Pollut. 157, 1140e1151.

Paolicelli, P., de la Fuente, M., Sánchez, A., Seijo, B., Alonso, M.J., 2009. Chitosannanoparticles for drug delivery to the eye. Expert Opin. Drug Deliv. 6, 239e253.

Park, K., Chen, Y., Hu, Y., Mayo, A.S., Kompella, U.B., Longeras, R., Ma, J.-X., 2009.Nanoparticle-mediated expression of an angiogenic inhibitor amelioratesischemia-induced retinal neovascularisation and diabetes-induced retinalvascular leakage. Diabetes 58, 1902e1913.

Peeters, L., Sanders, N.N., Braeckmans, K., Boussery, K., van de Voorde, J., deSmedt, S.C., Demeester, J., 2005. Vitreous: a barriers to nonviral ocular genetherapy. Invest. Ophthalmol. Vis. Sci. 46, 3553e3561.

Pierscionek, B.K., Li, Y., Yasseen, A.A., Colhoun, L.M., Schachar, R.A., Chen, W., 2010.Nanoceria have no genotoxic effect on human lens epithelial cells. Nanotech-nology 21, 035102.

Pignatello, R., Bucolo, C., Ferrara, P., Maltese, A., Puleo, A., Puglisi, G., 2002. EudragitRS100 nanosuspensions for the ophthalmic controlled delivery of ibuprofen.Eur. J. Pharm. Sci. 16, 53e61.

Pinto-Reis, C., Neufeld, R.J., Ribeiro, A.J., Veiga, F., 2006. Nanoencapsulation I.Methods for preparation of drug-loaded polymeric nanoparticles. Nano-medicine 2, 8e21.

Proske, D., Blank, M., Buhmann, R., Resch, A., 2005. Aptamers-basic research, drugdevelopment, and clinical applications. Appl. Microbiol. Biotechnol. 69,367e374.

Prow, T.W., 2009. Toxicity of nanomaterials to the eye. WIREs Nanomed. Nano-biotechnol. Dec 11.

Putman, E., van der Laan, J.W., van Loveren, H., 2003. Assessing immunotoxicity:guidelines. Fundam. Clin. Pharmacol. 17, 615e626.

Qaddoumi, M.G., Gukasyan, H.J., Davda, J., Labhasetwar, V., Kim, K.J., Lee, V.H.L.,2003. Clathrin and caveolin-1 expression in primary pigmented rabbitconjunctival epithelial cells: Role in PLGA nanoparticle Endocytosis. Mol. Vis. 9,559e568.

Quigley, H.A., Broman, A.T., 2006. The number of people with glaucoma worldwidein 2010 and 2020. Br. J. Ophthalmol. 90, 262e267.

Rabinowitz, J.E., Samulski, J., 1998. Adeno-associated virus expression systems forgene transfer. Curr. Opin. Biotechnol. 9, 470e475.

Ralph, G.S., Binley, K., Wong, L.F., Azzouz, M., Mazarakis, N.D., 2006. Gene therapyfor neurodegenerative and ocular diseases using lentiviral vectors. Clin. Sci.(Lond) 110, 37e46.

Reddy, S.T., van der Vlies, A.J., Simeoni, E., Angeli, V., Randolph, G.J., O ’Neil, C.P.,Lee, L.K., Swartz, M.A., Hubbell, J.A., 2007. Exploiting lymphatic transport andcomplement activation in nanoparticle vaccines. Nat. Biotechnol. 25,1159e1164.

Roy, K., Stein, L., Kaushal, S., 2010. Ocular gene therapy: an evaluation of recombinant adeno-associated virus-mediated gene therapy interventions forthe treatment of ocular disease. Hum. Gene Ther. 21, 915e927.

Rzigalinski, B.A., Bailey, D., Chow, L., Kuiry, S.C., Patil, S., Merchant, S., et al., 2003.Cerium oxide nanoparticles increase the lifespan of cultured brain cells andprotect against free radical and mechanical trauma. FASEB J. 17, A606.

Sahoo, S.K., Dilnawaz, F., Krishnakumar, S., 2008. Nanotechnology in ocular drugdelivery. Drug Discov. Today (3/4), 144e151.

Sahoo, S.K., Parveen, S., Panda, J.J., 2007. The present and future of nanotechnologyin human health care. Nanomedicine 3, 20e31.

Sakai, T., Kohno, H., Ishihara, T., Higaki, M., Saito, S., Matsushima, M., Mizushima, Y.,Kitahara, K., 2006. Treatment of experimental autoimmune uveoretinitis withpoly(lactic acid) nanoparticles encapsulating betamethasone phosphate. Exp.Eye Res. 82, 657e663.

Sánchez, A., Alonso, M.J., 2006. Nanoparticular carriers for ocular drug delivery. In:Torchilin, Vladimir (Ed.), Nanoparticulates as Drug Carriers. World Scientific-Imperial College Press, ISBN 1-86094-630-5, pp. 1e30 (Chapter 27).

Sasaki, H., Yamamura, K., Mukai, T., Nishida, K., Nakamura, J., Nakashima, M.,Ichikawam, M., 1999. Enhancement of ocular drug penetration. Crit. Rev. Ther.Drug Carrier Syst. 16, 85e146.

Schoenwald, R.D., 1990. Ocular drug delivery: pharmacokinetics considerations.Clin. Pharmacokinet. 18, 255e269.

Snodin, D.J., 20 04. Regulatory immunotoxicology: does the published evidencesupport mandatory nonclinical immune function screening in drug develop-ment? Regul. Toxicol. Pharmacol. 40, 336e355.

Snyder, R.O., 1999. Adeno-associated virus-mediated gene delivery. J. Gene Med. 1,166e175.

Soppimath, K.S., Aminabhavi, T.M., Kulkarni, A.R., Rudzinski, W.E., 2001. Biode-gradable polymeric nanoparticles as drug delivery devices. J. Control Release 70,1e20.

Stone, V., Johnston, H., Schins, R.P., 2009. Development of in vitro systems fornanotoxicology: methodological considerations. Crit. Rev. Toxicol. 39, 613e626.

Szebeni, J., Alving, C.R., Rosivall, L., Bünger, R., Barany, L., Bedöcs, P., Tóth, M.,Barenholz, Y., 2007. Animal models of complement-mediated hypersensitivityreactions to liposomes and other liposome-based nanoparticles. J. LiposomeRes. 17, 107e117.

Tarnuzzer, R.W., Colon, J., Patil, S., Seal, S., 2005. Vacancy engineered ceria nano-structures for protection from radiation-induced cellular damage. Nano Lett. 12,2573e2577.

Tanelian, D.L., Barry, M.A., Johnston, S.A., Le, T., Smith, G., 1997. Controlled gene gundelivery and expression of DNA within the cornea. Biotechniques 23, 484e488.

Toyama, T., Matsuda, H., Ishida, I., Tani, M., Kitaba, S., Sano, S., Katayama, I., 2008.A case of toxic epidermal necrolysis-like dermatitis evolving from contactdermatitis of the hands associated with exposure to dendrimers. Contact Derm.59, 122e123.

Urtti, A., 2006. Challenges and obstacles of ocular pharmacokinetics and drugdelivery. Adv. Drug Deliv. Revs. 58, 1131e1135.

Vanderwoot, J., Ludwig, A., 2007. Ocular drug delivery: nanomedicines application.Nanomedicine 2, 11e21.Vega, E., Egea, M.A., Valls, O., Espina, M., García, M.L., 2006. Flurbiprofen loaded

biodegradable nanoparticles for ophthalmic administration. J. Pharm. Sci. 95,2393e2405.

Wadhawa, S., Paliwal, R., Paliwal, S.R., Vyas, S.P., 2009. Hyaluronic acid modified chi-tosan nanoparticles for effective management of glaucoma: development, char-acterization, and evaluation. J. Drug Target.. doi:10.3109/10611860903450023Nov 27 (Epub ahead of print).

Wood, R.W., Lee, H.K., Kreuter, J., Robinson, J.R., 1985. Ocular disposition of poly-hexyl-2-cyano(3-14C)acrylate nanoparticles in the albino rabbit. Int. J. Pharm.23, 175e183.

Xu, J., Heyes, J.J., Barocas, V.H., Randolph, T.W., 2000. Permeability and diffusion invitreous humour: implications for drug delivery. Pharm. Res. 17, 664e669.

Yamamoto, S., Manabe, N., Fujioka, K., Hoshino, A., Yamamoto, K., 2007. Visualizingvitreous using quantum dots as imaging agents. IEEE Trans. Nanobioscience 6,94e98.

Yanagisawa, R., Takano, H., Inoue, K.I., Koike, E., Sadakane, K., Ichinose, T., 2009.Titanium dioxide nanoparticles aggravate atopic dermatitis-like skin lesions in

NC/Nga mice. Exp. Biol. Med. 234, 314e

322.Yanagisawa, R., Takano, H., Inoue, K.I., Koike, E., Sadakane, K., Ichinose, T., 2010. Size

effects of polystyrene nanoparticles on atopic dermatitis-like skin lesions inNC/NGA mice. Int. J. Immunopathol. Pharmacol. 23, 131e141.

Yenice, I., Mocan, M.C., Palaska, E., Bochot, A., Bilensoy, E., Vural, I., Irkeç, M.,Hincal, A.A., 2008. Hyaluronic acid coated poly-epsilon-caprolactone nano-spheres deliver high concentrations of cyclosporine A into the cornea. Exp. EyeRes. 87, 162e167.

Zavaglia, D., Normand, N., Brewis, N., O ’Hare, P., Favrot, M.C., Coll, J.L., 2003. VP22-mediated and light-activated delivery of anti-c-raf1 antisense oligonucleotideimproves its activity after intratumoral injection in nude mice. Mol. Ther. 8,840e845.

Ziche, M., Donnini, S., Morbidelli, L., 2004. Development of new drugs in angio-genesis. Curr. Drug Targets 5, 485e4493.

Zimmer, A., Kreuter, J., Robinson, J.R., 1991. Studies on the transport pathway of PBCA nanoparticles in ocular tissues. J. Microencapsul. 8, 497e504.

Zimmer, A., Mutschler, E., Lambrecht, G., Mayer, D., Kreuter, J., 1994. Pharmacoki-netic and pharmacodynamic aspects of an ophthalmic pilocarpine nano-particle-delivery-system. Pharm. Res. 11, 1435e1442.

Zolnik, B.S., González-Fernández, A., Sadrieh, N., Dobrovolskaia, M.A., 2010.Minireview: nanoparticles and the immune system. Endocrinology 151,458e465.


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