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Nanotoxicology
ISSN: 1743-5390 (Print) 1743-5404 (Online) Journal homepage: http://www.tandfonline.com/loi/inan20
Safety and toxicity of nanomaterials for oculardrug delivery applications
Neelesh K. Mehra, Defu Cai, Lih Kuo, Travis Hein & Srinath Palakurthi
To cite this article: Neelesh K. Mehra, Defu Cai, Lih Kuo, Travis Hein & Srinath Palakurthi(2016): Safety and toxicity of nanomaterials for ocular drug delivery applications,Nanotoxicology
To link to this article: http://dx.doi.org/10.3109/17435390.2016.1153165
Published online: 30 Mar 2016.
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http://informahealthcare.com/nanISSN: 1743-5390 (print), 1743-5404 (electronic)
Nanotoxicology, Early Online: 1–25! 2016 Taylor & Francis. DOI: 10.3109/17435390.2016.1153165
REVIEW ARTICLE
Safety and toxicity of nanomaterials for ocular drug deliveryapplications
Neelesh K. Mehra1, Defu Cai1, Lih Kuo2,,3, Travis Hein3and Srinath Palakurthi1,
1Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M Health Science Center, Kingsville, TX, USA,2Department of Medical Physiology, College of Medicine, Texas a&M Health Science Center, Temple, TX, USA, 3Department of Surgery and Scott &
White Eye Institute, College of Medicine, Texas a&M Health Science Center, Temple, TX, USA
Abstract
Multifunctional nanomaterials are rapidly emerging for ophthalmic delivery of therapeutics tofacilitate safe and effective targeting with improved patient compliance. Because of theirextremely high area to volume ratio, nanomaterials often have physicochemical properties thatare different from those of their larger counterparts. There exists a complex relationshipbetween the physicochemical properties (composition, size, shape, charge, roughness, andporosity) of the nanomaterials and their interaction with the biological system. The eye is a verysensitive accessible organ and is subjected to intended and unintended exposure tonanomaterials. Currently, various ophthalmic formulations are available in the market, whilesome are underway in preclinical and clinical phases. However, the data on safety, efficacy, andtoxicology of these advanced nanomaterials for ocular drug delivery are sparse. Focus of thepresent review is to provide a comprehensive report on the safety, biocompatibility andtoxicities of nanomaterials in the eye.
Keywords
Carbon nanomaterials, eye, nanomaterials,nanoparticles, safety and toxicity
History
Received 23 December 2015Revised 5 February 2016Accepted 8 February 2016Published online 25 March 2016
Introduction
Nanotechnology has been a rapidly expanding field and iscontinuously being explored in the field of pharmaceutical andbiotechnology sciences to achieve the desired product or thera-peutic outcomes. Nanotoxicology is a branch of nanoscience thatdeals with the safety and toxicity of nanomaterials and evaluatingtheir interactions with biological systems. Due to the nano-size andhigh surface free area, these nanomaterials interact with the cellsand subcellular organelles rapidly and effectively, which may leadto toxic effects. The nanotechnology industry is a rapidly emergingindustry generating numerous engineered nanomaterials (ENM)that are incorporated into medical, cosmetic and consumerproducts. Thus far, more than 3000 nanomaterials have beenused in pharmaceutical companies, but toxicity data are sparse(Jain et al., 2007, 2014; Mody et al., 2014; Srivastava et al., 2015).The widespread production and use of ENM may lead tounintended human exposure, while their impact on biologicalsystems is not clearly understood. A comprehensive understandingof the nano-bio interactions would not only facilitate developingsafe and efficacious nanoproducts for therapeutic applications, butit also would aid in developing guidelines for the propermanufacturing, production, synthesis, handling, use and disposalof nanomaterials. In addition to the users of nanoproducts such ascosmetics and other consumer products, personnel involved inmanufacturing, handling, and working on nanomaterials are at riskof exposure to the nanomaterials by contact (skin and eye),
inhalation (nose and lung), and ingestion (gastrointestinal tract). Toaddress these concerns, the National Institute of EnvironmentalHealth Sciences (NIEHS) initiated a NanotechnologyEnvironmental Health and Safety (NanoEHS) program in 2004 togain fundamental understanding of the molecular and pathologicalpathways involved in mediating biological responses to ENM.
Currently, various ophthalmic formulations are available in themarket, while some are underway in preclinical and clinical phases.Herein, we attempted to present a comprehensive review on thesafety, biocompatibility and toxicity of nanomaterials in the eye.The presence of various anatomical and physiological barriers is amajor constraint on ophthalmic drug delivery, which prevents thedrug from reaching the target site. Topically applied drugs canenter into the anterior chamber by crossing the cornea or throughthe conjunctiva and sclera or via the systemic circulation, but theymust cross the blood-aqueous barrier. As a recent report suggests,topically applied ophthalmic eye drops typically deliver drugs intothe anterior chamber and negligible amounts (55%) enter into theback of the eye (Fitzpartrick et al., 2012).
In ophthalmic delivery, conventional ophthalmic formulationsare easily washed away by nasolacrimal drainage, while thenanoparticles are cleared slowly and release the drug for a longerduration upon interaction with the cornea. These nanomaterialsare expected to not only enhance the ocular bioavailability butalso offer sustained therapeutic drug concentration for a longerduration and thereby reduce frequency of administration withimproved patient compliance.
Basic structure of the eye
The eye is a complex, sensitive organ with numerous absorptionbarriers. It is composed of three main layers: the sclera and cornea
*Correspondence: Srinath Palakurthi [email protected] of Pharmaceutical Sciences, Irma Lerma Rangel College ofPharmacy Texas A&M Health Science Center, Kingsville, TX 78363,USA
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(outer layer); the choroid, ciliary body, and iris (middle layer oruveal coat); and the retina (inner layer). The outer part of the eyeis mainly covered by the mobile tarsal part of eyelids. The eyeballis covered by the thin skin of lids, which permits opening andclosing of the palpebral fissure. For spontaneous blinking, eyesare under voluntary and involuntary control. Generally, thera-peutics penetrate the corneal epithelium by either paracellular ortranscellular transport mechanisms. The transcellular mechanismpredominates for lipophilic molecules, while hydrophilic mol-ecules and ions are transported by a paracellular route. The poresize of the corneal epithelium is in the range of 1–5 nm (Prausnitz& Noonan, 1998). Only about 5% of the topically administereddrug is absorbed through the corneal epithelium. The cornea is aclear, transparent, avascular tissue and it is composed ofepithelium, stroma, Descemet’s membrane, Bowman’s layer, andendothelium. The corneal epithelium consists of 5–6 layers ofcolumnar cells squeezed forward by the new cells. The cornealepithelial cell surface is susceptible to fungal, bacterial, and viralinfections and inflammation. Replacement of the epithelial cellsoccurs by mitotic division of the basal layer every 4–8 d. Thelachrymal fluid and the aqueous humor supply nutrients andoxygen to the cornea. Fungal infection of the cornea is mainlycaused by the fungal species of Aspergillus, Fusarium,Curvularia, and Candida, which may lead to cataract, loss ofvision, and blindness. The conjunctiva is a thin transparentmembrane, which lines the inner surface of eyelids. Theconjunctiva is composed of an epithelium, a highly vascularizedsubstantia propria, and a submucosa or episclera (Kim et al.,2015; Sosnik et al., 2014; Yan et al., 2012). The sclera is a fibrouslayer composed of collagen and elastic fibers. It offers resistanceto internal and external forces and provides mechanical stabilityto the eye. The scleral surface area in humans is approximately16–17 cm2 and largely influences the pharmacokinetics of drugdiffusion (Myles et al., 2005). The sclera is 10 times morepermeable than the cornea and the conjunctiva is 15–25 timesmore permeable than the sclera (Hamalainen et al., 1997). Poorcorneal penetration, short precorneal residence time, and presence
of various anatomical and physiological barriers are the mainproblems in ocular therapeutic delivery. Additionally, the blood–retinal barrier, the blood–aqueous barrier, and the extraocularepithelium represent obstacles in ocular delivery to the choroid,retina, and vitreous. Only a fraction of the topically administereddrug reaches the retina or the vitreous body following systemicadministration (Kim et al., 2015; Lezzi et al., 2012). Directintravitreal injection is performed to achieve high drug concen-trations in the vitreous and retina. However, frequent injectionsare required to sustain high drug levels because the half-life ofdrugs in the vitreous cavity is relatively short. Nanoparticlesystems that can provide sustained drug release have beenexplored to reduce the frequency of intravitreal administration.Figure 1 shows various distribution pathways for administration ofocular drugs in the eye.
Parameters to assess toxicity to the eye
There are various observable and measurable parameters fortesting the toxicity of ophthalmic formulations. A comprehensivereview on the techniques currently used for ocular toxicity testingis published by Wilson et al. (2015). While a myriad ofnanomaterials are used in FDA-regulated ocular products,immense data gaps, and conflicting reports on their toxicologycurrently prevent generalizing how nanomaterial physicochemicalproperties relate to biological activity and toxic potential (Jainet al., 2014; Srivastava et al., 2015). Moreover, little is knownabout the interaction of nanomaterials with the cells andpenetration/toxicity.
The international standard assay for acute ocular toxicity (eyeirritation test) is the rabbit in vivo Draize eye test, developed bythe FDA (Draize et al., 1994). The Draize eye irritation test is anendorsed method/assay to validate the safety and toxicity ofmaterials being used in ophthalmic preparations. Followingtopical exposure of nanomaterials (100 mL or 100 mg) to theconjunctival sac for 72 h, the rabbits are observed up to 21 d atregular intervals for signs of irritation (observable toxicity)
Figure 1. Various distribution pathways for administration of ocular drugs in the eye.
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including redness, swelling, cloudiness, edema, hemorrhage,discharge, and blindness (Huhtala et al., 2008). New Zealandwhite (NZW) rabbits are most widely used model because theyhave large eyes with known anatomy and physiology, easy tohandle, available and inexpensive. Eye irritation is summarized asa ‘‘maximum average score’’ (MAS), which is an average valuescored based on the corneal injury for individual animals at thetime of scoring. Despite its gold standard status, the Draize testwas never formally validated. Moreover, since the anatomy,physiology, biochemistry of the rabbit eye differs from the humaneye (thinner cornea, lower tear production, blinking frequency andocular surface sensitivity in rabbits), differences in sensitivity toirritants can occur. Rabbits have larger conjunctival sacs and anictitating membrane, which may aid in removal of the testsubstance from the ocular surface.
The low-volume eye-irritation test (LVET) is refinement of theDraize test and was developed by Griffith et al. (1980). In theLVET, 0.01 ml/0.01 g lower volume of test substances is appliedinto the right eye of animals. The LVET test is less stressful to theanimal than the Draize test (Roggeband et al., 2000). It ischaracterized by changes in the pathological condition of cornea,conjunctiva, and iris/ciliary body. Recently, the InteragencyCoordinating Committee on the Validation of AlternativeMethods (ICCVAM) did not consider nor recommend forprospective ocular safety testing test in replacement of Draizetesting. In our opinion, we can consider the LVET toxicity testingmethod as an alternative eye toxicity test.
Ocular organotypic models or the enucleated eye test (EET)uses isolated rabbit eyes from animals used for other research orcommercial purposes (Barile, 2010). The nanomaterials may beapplied at relevant concentrations and corneal opacity andswelling are observed. Corneal opacity is an indication of proteindenaturation, swelling, vacuolation, and damage of epitheliumand stroma. Bovine and porcine cornea and chicken enucleatedeyes are widely accepted as a reliable and an accurate tissue forassessing eye irritation. EET is considered a scientifically soundtest to evaluate and identify substances that are non-irritants aswell as those materials that cause irreversible eye damage.
The test materials (including nanomaterials and formulation)that penetrate the ocular tissues or that are administeredintraocularly may show some impact on the retinal morphologyand circulation, which may experimentally be assessed usingoptical coherence tomography (OCT), ultrasound Doppler meas-urements and retinal fluorescein angiography, respectively. TheOCT is used to assess architectural changes in the nerve fiber andganglion cell layers, inner retina, photoreceptor cell layer, retinalthickness, choroidal thickness, and the vitreoretinal interface(including the development of epiretinal membranes and vitreo-macular traction) (Kjellstrom et al., 2014; Majdi et al., 2015; vanVelthoven et al., 2007). The effect of nanomaterials on ophthal-mic blood flow, which is controlled by the downstream arteriolesin the microcirculation, may be assessed using color Dopplerultrasound (Anayol et al., 2014; Dong et al., 2000; Plange et al.,2003). To detect whether exposure of test material in the eyecould cause vascular changes, such as focal constriction/dilationof the local vasculature, retinal fluorescein angiography can beperformed (Dong et al., 2000; Plange et al., 2003). To correlatethe structural and vascular changes observed in the above threetests to the neural function of the retina, toxicity to the retinalneurons can by assessed by electroretinography (ERG) (Huanget al., 2015; Kjellstrom et al., 2014). The ERG measures theelectrical and non-electrical activities generated by neural andnon-neuronal cells in the retina in response to a light stimulusfollowing exposure to the test material (Huang et al., 2015;Kjellstrom et al., 2014). Although these experimental tests are notyet validated by the regulatory agencies, they offer detailed
information on any changes in vascular and neural structure andfunctions following exposure to the test material, and guide in thedevelopment of safe ocular formulations and next generationnanomaterials.
Nanomaterials-mediated eye therapy
Several site-specific ocular therapeutic delivery systems havebeen evaluated so far, including carbon nanohorns (Miyawakiet al., 2008), fullerenes (Aoshima et al., 2009), graphenes(Angelopoulou et al., 2015; Mao et al., 2013; Yan et al., 2012),quantum dots (Kuo et al., 2011), carbon nanotubes (CNTs) (Luet al., 2013), contact lenses (Kapoor et al., 2009; Wu et al., 2015),hydrogels (Almeida et al., 2014; Kirchhof et al., 2015;Sheikholeshlami et al., 2015), corneal SkGel (SkGel implantfrom corneal laboratory) (Lopes et al., 2006), chitosan (Mannaet al., 2015), polymeric micelles (Tommaso et al., 2011),dendrimers (Holden et al., 2012; Lezzi et al., 2012; Souzaet al., 2015; Spataro et al., 2010), implants (Bozukova et al.,2010), triblock copolymers (Qiao et al., 2013), liposomes (Caiet al., 2014; Chetoni et al., 2015; Taha et al., 2014), niosomes(Marianecci et al., 2014), nanoparticles (Abrego et al., 2015;Andrei et al., 2015; Leonardi et al., 2014; Soderstjerna et al.,2014), micro- and nano-emulsions (Gan et al., 2009), nanosus-pensions (Pignatello et al., 2002), nanoaggregates (Dilbaghi et al.,2013), nanocrystals (Tuomela et al., 2014), micro-needles (Kimet al., 2015), cyclodextrins (Garcia-Fernandez et al., 2013; HalimMohamed & Mahmoud, 2011), and electric-pulse targeted(Oshima et al., 1999). The nanomaterials that have been exploredfor ocular drug delivery are illustrated in Figure 2.
Ophthalmic delivery to anterior or posterior segments of theeye has been investigated using a variety of drugs, includinghomoharringtonine (HHT) (Peng et al., 1999), mitomycin C(Kozobolis et al., 2002), autumn narcissus alkali (Fuller et al.,2002), human amniotic membrane (Rai et al., 2005), tissueplasminogen activator (Tripathi & Tfipathi, 2005), paclitaxel(Koz et al., 2007), corticosteroids (Spitzer et al., 2008), 5-fluorouracil (Wang et al., 2011), cyclosporine A (Aksungur et al.,2011; Gan et al., 2009), interferons (Wang et al., 2011), aspirin(Das et al., 2012), natamycin (Bhatta et al., 2012), timololmaleate, brimonidine (Yang et al., 2012), terbinafine hydrochlor-ide (Tayel et al., 2013), tropicamide (Dilbaghi et al., 2013),pilocarpine nitrate (Li et al., 2013), indomethacin (Nagai et al.,2014), acetazolamide (Mishra & Jain, 2014), brinzolamide(Tuomela et al., 2014), dorzolamide (Warsi et al., 2014),distamycin (Chetoni et al., 2015), and dexamethasone (Courseyet al., 2015). For the reader’s interest, Kompella et al. (2013)published a review of various nanomedicines used for delivery oftherapeutics into the back of the eye entitled ‘‘Nanomedicines forback of the eye drug delivery, gene delivery, and imaging’’.
Nanotoxicology: safety and toxicity of nanomaterials
Although a significant amount of data is available on theformulation, characterization, ocular drug delivery and targetingof nanomaterials, information on the safety and toxicity of theabove mentioned delivery systems and multifunctional nanoma-terials is sparse. Safety and toxicity are important issues that arealways of primary concern prior to approval of ophthalmicproducts for clinical trials. Although in-depth toxicology studiesof nanomaterials to the eye were summarized by Prow (2010),herein we attempt to present a detailed review on the safety andtoxicological perspectives of new nanomaterials and deliverysystems that are under various phases of preclinical and clinicalphases. Table 1 summarizes the safety and toxicity of nanomater-ials in the eye.
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Nanoparticles
Nanoparticles are colloidal particles having at-least one dimen-sion less than 100 nm (Mehra et al., 2015b; Rajala et al., 2014).Decrease in the particle size causes less irritation allows for ahigher surface to volume ratio, increases precorneal residencetime, and minimizes drug dose and chances of toxicity more thanmicroparticles (Das et al., 2012). Corneal toxicity and thepropensity of the drug to penetrate and permeate across thecornea increased with using smaller size nanoparticles (Conley &Naash, 2010). The tranilast nanoparticles dispersion, which wasprepared using the bead mill method, caused less damage tohuman corneal epithelium (HCE) cells. The nanoparticles alsoenhanced the corneal penetration as compared with tranilast eyedrops (RIZABEN� eye drops) (Nagai & Ito, 2014; Nagai et al.,2014).
Pilocarpine nitrate (PN) has been used in the treatment of acuteangle-closure and chronic open-angle glaucoma. However, itshows poor corneal penetration and low pre-ocular retention, andtherefore poor bioavailability. Due to the poor ocular bioavail-ability, it requires frequent administration in high concentrations,leading to side effects (myopia and miosis). Liquid crystallinenanoparticles (LCNPs) offer some unique properties like highinternal interfacial area useful for encapsulating both hydrophobicand hydrophilic as well as amphiphilic drugs in separate domains.LCNPs are a better nano-candidate for ocular delivery, whereinPNs are well entrapped. Additionally, it is non-toxic, bioadhesivein nature and depicts sustained-release behavior. Glycerylmonoolein (GMO) is a non-toxic, biocompatible and biodegrad-able amphiphilic lipid that swells in the water and thenspontaneously forms a well-ordered liquid crystalline phase (Liet al., 2013; Nair et al., 2012).
Li and et al. (2013) investigated the potential of GMO-basedLCNPs for ocular delivery of PNs (PN-LCNPs) (particle size202.28 ± 19.32 nm and percent encapsulation efficiency 61.03%)in adult New Zealand white rabbits. From the ex vivo corneal
permeation study, PN-LCNPs treatment led to a two-fold higherapparent permeability coefficient than did commercial eye dropsand decreased IOP in vivo. PN-LCNPs did not cause irritation ofocular tissues in the rabbit eye based on the conjunctival scores.
Dorzolamide (Trusopt�) is a potent and frequently prescribed
drug in the treatment of glaucoma. But dorzolamide has poorocular retention and its degree of ionization and hydrophilicnature leads to poor ocular bioavailability. It is used three to fourtimes a day. Dorzolamide-loaded PLGA nanoparticles using twoemulsifiers poly (vinyl alcohol) (PVA) and tocopheryl polyethyl-ene glycol succinate (TPGS) for glaucoma therapy weredeveloped and reported in freshly excised goat eyes and adultNew Zealand albino rabbits by Warsi et al. (2014). Ex vivo trans-corneal permeation study and pharmacoscintigraphic studiesrevealed that the PVA and TPGS nanoparticles are non-irritatingand significantly reduce the intraocular pressure by 22.81 and29.12%, respectively, via topical instillation.
Azone is one of the most widely used permeation enhancers toimprove the permeability of both hydrophilic and lipophilic drugs.Recently, the biopharmaceutical profile of pranoprofen-loadedPLGA nanoparticles (PF-F1NPs and PF-F2NPs) were developedand further dispersed into hydrogels for ocular administration.The optimized PF-NP suspensions were dispersed in freshlyprepared carbomer hydrogels (HG_PF-F1NPs and HG_PF-F2NPs) or in hydrogels containing 1% azone (HG_PF-F1NPs-Azone and HG_PF-F2NPs-Azone) to increase the ocular bio-pharmaceutical profile of pranoprofen. It is a non-steroidal anti-inflammatory drug used as a safe and effective alternative anti-inflammatory treatment following strabismus and cataract sur-gery. No signs of ocular irritancy have been observed with thehydrogels based on the in vitro and in vivo ocular tolerance andin vivo anti-inflammatory studies on New Zealand rabbits(male, weighing 2.5–3.0 kg). The ocular application of hydrogelscontaining azone was more effective than azone-free formulationsin the treatment of edema on the ocular surface (Abrego et al.,2015).
Figure 2. Nanomaterials used in ocular delivery of therapeutic agents.
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Tab
le1
.S
um
mar
ize
the
safe
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om
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Tab
le1
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on
tin
ued
Nan
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ith
nan
oem
uls
ion
show
edcl
eare
rh
eal-
ing
of
corn
eal
chem
ical
ulc
erw
ith
mo
der
ate
effe
ctiv
ein
hib
itio
no
fp
oly
mo
rph
nu
clea
rle
uck
-o
cyti
cin
filt
ra-
tio
n(P
MN
Ls)
com
par
edw
ith
nan
op
ar-
ticl
esp
rep
arat
ion
Bad
awi
etal
.(2
00
8)
Ch
ito
san
hyla
uro
nic
acid
Hu
man
corn
eal
epit
hel
ial
(HC
E)
and
no
rmal
hu
man
con
jun
ctiv
al(I
OB
A-N
HC
)ce
lls
No
tp
erfo
rmed
pE
GF
Po
rp
b-g
alN
ot
per
form
edN
on
tox
icC
orn
eaH
A-C
HI
nan
o-
par
ticl
estr
ansf
ergen
esto
the
ocu
lar
surf
ace
thro
ug
hC
D4
4re
cep
tor
med
iate
dfl
uid
end
ocy
tosi
s
De
laF
uen
teet
al.
(20
08
)
Ch
ioto
san
and
mag
net
icn
ano
par
ticl
es
Ret
inal
pig
men
tep
ith
elia
lce
lls
New
Zea
lan
dw
hit
ean
dD
utc
hB
elte
dra
bb
its
Gen
eIn
trav
itre
ally
(IV
)o
rsu
bre
-ti
nal
lly
(SR
)
No
tin
flam
ma-
tory
and
did
no
tin
du
cere
tin
alp
ath
olo
gy
Ret
ina
Go
od
tran
sfec
-ti
on
wit
hle
ssto
xic
ity.
Pro
wet
al.
(20
08
)
(co
nti
nu
ed)
6 N.K. Mehra et al. Nanotoxicology, Early Online: 1–25
Dow
nloa
ded
by [
Tex
as A
&M
Uni
vers
ity L
ibra
ries
] at
06:
58 3
1 M
arch
201
6
Nan
om
ater
ials
Ex
vivo
stu
dy
Invi
vost
ud
yT
her
apeu
tic
del
iver
edR
ou
tes
of
adm
inis
trat
ion
Tox
icit
yT
arget
site
Ou
tco
me
Ref
Go
ldN
ano
par
ticl
es(G
NP
s)
Hu
man
corn
eain
vitr
oR
abb
itco
rnea
sin
vivo
PE
I2-g
ene
To
pic
alro
ute
Low
-tox
icC
orn
eal
tiss
ue
PE
I2-G
NP
sar
elo
wto
xic
,ra
pid
up
take,
and
slow
clea
ran
cean
du
sefu
lfo
rgen
eth
erap
yin
vivo
Sh
arm
aet
al.
(20
11
)
Sil
ver
and
go
ldn
ano
par
ticl
esC
ult
ure
dp
ost
-n
atal
mo
use
reti
na.
C3
Hw
ild
-ty
pe
(wt)
mic
eN
on
eT
ox
icN
P-r
elat
edn
euro
nal
tox
icit
y
Ret
ina
low
con
cen
tra-
tio
ns
of
20
and
80
nm
size
dA
g-
and
Au
NP
sh
ave
adver
seef
fect
so
nth
ere
tin
a,u
sin
gan
org
ano
ty-
pic
reti
na
cul-
ture
mo
del
So
der
stje
rna
etal
.(2
01
4)
PL
GA
nan
op
arti
cles
–N
ewZ
eala
nd
Rab
bit
sP
ran
op
rofe
nE
xv
ivo
corn
eal
per
mea
tio
nst
ud
y
No
tox
icit
yw
asse
enC
orn
eaM
ore
effe
ctiv
ein
ocu
lar
ther
apy
con
tain
ing
azo
ne
Ab
reg
oet
al.
(20
15
)
PA
MA
Md
end
ri-
mer
sse
ries
wit
h-C
OO
Han
d–
OH
fun
ctio
nal
gro
up
s
–N
ewZ
eala
nd
alb
ino
Rab
bit
sP
ilo
carp
ine
nit
rate
and
tro
pic
amid
e–
––
Pro
lon
ged
dru
gre
sid
ence
tim
eV
and
amm
ean
dB
rob
eck
(20
05
)
5.0
GP
PI
den
dri
mer
sN
ot
per
form
edN
ewZ
eala
nd
alb
ino
Rab
bit
sA
ceta
zola
mid
e(A
CZ
)L
ow
ercu
l-d
e-sa
co
fra
bb
its
eye
Hem
oly
tic
tox
icit
y–
AC
Z-l
oad
edP
PI
den
dri
mer
sen
han
ces
ocu
lar
bio
-av
aila
bil
ity,
ocu
lar
resi
-d
ence
tim
e(c
ul-
de
sac)
of
AC
Zw
ith
less
ocu
lar
irri
tan
cy
Mis
hra
and
Jain
(20
14
)
Hy
dro
xyl-
term
i-n
ated
PA
MA
Md
end
rim
ers
–H
om
ozy
go
us
rece
ssiv
erd
yRoyal
Co
lleg
eo
fS
urg
eon
sal
bin
ora
ts(p
ron
eto
ret-
inal
Flu
oci
no
lon
eace
ton
ide
Intr
avit
real
Ou
ter
reti
na
Low
erd
ose
of
dru
gan
dle
sssi
de
effe
cts
and
clea
red
inta
ct
Lez
ziet
al.
(20
12
)
(co
nti
nu
ed)
DOI: 10.3109/17435390.2016.1153165 Please check 7
Dow
nloa
ded
by [
Tex
as A
&M
Uni
vers
ity L
ibra
ries
] at
06:
58 3
1 M
arch
201
6
Tab
le1
.C
on
tin
ued
Nan
om
ater
ials
Ex
vivo
stu
dy
Invi
vost
ud
yT
her
apeu
tic
del
iver
edR
ou
tes
of
adm
inis
trat
ion
Tox
icit
yT
arget
site
Ou
tco
me
Ref
deg
ener
atio
n)
and
Sp
rag
ue
Daw
ley
rats
Ph
osp
ho
rou
sco
nta
inin
gd
end
rim
ers
–R
abb
its
Car
teo
lol
––
–S
pat
aro
etal
.(2
01
0)
Nan
ocr
yst
als
(hy
dro
xy
pro
pyl
met
hylc
ellu
lose
(HP
MC
),p
olo
xa-
mer
F1
27
and
F6
8,
po
lyso
rbat
e8
0)
Hu
man
corn
eal
epit
hel
ial
cell
(HC
E)
Sev
en-m
on
tho
ldm
ale
Wis
tar
rats
Bri
nzo
lam
ide
(BR
A)
To
pic
alN
o-t
ox
icE
ye
Th
ein
vivo
rat
ocu
lar
hy
per
-te
nsi
on
mo
del
show
edth
esi
gnif
ican
tly
dec
reas
edin
trao
cula
rp
ress
ure
val
ues
of
all
form
ula
tio
ns
Tu
om
ela
etal
.(2
01
4)
P1
23
/TP
GS
mix
edm
icel
les
insi
tugel
s(C
UR
-MM
-IS
Gs)
Ex
viv
oco
rnea
New
Zea
lan
dR
abb
itC
urc
um
inU
sin
gF
ran
z-d
if-
fusi
on
cel
lB
ioco
mp
atib
leC
orn
eaF
rom
exvi
voco
rnea
lp
en-
etra
tio
nst
ud
yin
dic
ated
that
the
cum
ula
-ti
ve
dru
gp
er-
mea
tio
nam
ou
nt
of
CU
R-M
M-
ISG
s(3
:1,
v/
v)
was
,re
spec
tivel
y,1
.16
-fo
ldan
d1
.32
-fo
ldh
igh
erth
anC
UR
-MM
-IS
Gs
(1:1
,v
/v
)an
dcu
rcu
-m
inso
luti
on
.
Du
anet
al.
(20
15
)
Met
hox
yp
oly
(eth
yle
ne
gly
col)
-hex
yls
ub
-st
itu
ted
po
ly(l
ac-
tid
e)m
icel
leca
rrie
rs
Imm
ort
aliz
edh
um
anco
r-n
eal
epit
hel
ial
(HC
E)
cell
s
Fem
ale
alb
ino
New
Zea
lan
dR
abb
it
Cycl
osp
ori
nA
(Cy
A)
To
pic
alN
on
tox
icE
ye
Go
od
tole
ran
ceU
sefu
lfo
rd
ryey
esy
nd
rom
e,au
toim
mu
ne
uvei
tis
and
pre
ven
tio
no
fco
rnea
lg
raft
inje
ctio
n
To
mm
aso
etal
.(2
01
1)
(co
nti
nu
ed)
8 N.K. Mehra et al. Nanotoxicology, Early Online: 1–25
Dow
nloa
ded
by [
Tex
as A
&M
Uni
vers
ity L
ibra
ries
] at
06:
58 3
1 M
arch
201
6
Nan
om
ater
ials
Ex
vivo
stu
dy
Invi
vost
ud
yT
her
apeu
tic
del
iver
edR
ou
tes
of
adm
inis
trat
ion
Tox
icit
yT
arget
site
Ou
tco
me
Ref
Su
rfac
tan
t;la
den
po
ly-(
2-h
yd
ro-
xyet
hyl
met
h-
acry
late
)p
-H
EM
Ahy
dro
gel
s
Rel
ease
kin
etic
sN
ot
per
form
edC
yA
NA
No
nto
xic
Eye
Bo
thB
rij
97
and
mic
roem
ul-
sio
n-l
aden
hy
do
rgel
sex
hib
itsl
ow
and
exte
nd
edC
yA
rele
ase
for
20
day
san
dco
ntr
oll
ed
Kap
oo
r&
Ch
ou
nan
(20
08
)
Sh
ear-
resp
on
sive
op
hth
alm
ichy
dro
gel
Hu
man
corn
eal
epit
hel
ial
(HC
E)
and
reti
nal
pig
-m
ent
epit
he-
lial
cell
s(A
RP
E1
9ce
lls,
po
ster
-io
rse
gm
ent)
New
Zea
lan
dW
hit
e(N
ZW
)ra
bb
its
–In
trav
itre
alN
oo
pac
ific
atio
no
fle
nes
,co
rnea
or
vit
rou
s,n
or
any
fun
ctio
nal
and
mo
rph
o-
log
ical
chan
ges
toth
ep
ost
erio
rse
g-
men
to
fey
e.
Ret
ina
and
corn
eaS
hea
r-re
spo
nsi
ve
op
hth
alm
ichy
dro
gel
show
sp
rom
is-
ing
po
ten
tial
ino
ph
thal
mic
del
iver
yin
bo
than
teri
or
and
po
ster
ior
seg
men
tas
wel
lv
itre
alre
pla
cem
ent.
Sh
eik
ho
lesh
lam
iet
al.
(20
15
)
MW
CN
Ts;
MW
CN
Ts-
OH
MW
CN
Ts-
CO
OH
Ret
inal
pig
men
tep
ith
eliu
m(R
PE
)ce
lls
No
tp
erfo
rmed
NA
NA
Car
box
yl-
MW
CN
Ts
are
no
tto
xic
toey
e
Co
rnea
Th
eca
rbox
yl-
MW
CN
Ts
show
bes
to
cula
rb
io-
com
pat
ibil
ity
and
less
tox
ic.
Lu
etal
.(2
01
3)
MW
CN
Ts
of
two
dif
fere
nt
len
gth
sE
gg
Ch
ori
on
All
anto
icM
emb
ran
e(H
E-C
AM
)
Eye
irri
tati
on
test
on
rab
bit
NA
Co
nju
nct
ival
sac
of
left
eye
No
nir
rita
nt
Eye
Invi
voo
cula
rte
stre
sult
ssu
gges
tth
atM
WC
NT
of
bo
thsi
zes
wer
em
inim
-al
lyir
rita
tin
gw
her
eas,
the
invi
tro
HE
-C
AM
resu
ltis
no
n-i
rrit
ant
Kis
ho
reet
al.
(20
09
)
Sin
gle
-wal
led
carb
on
nan
oh
orn
s
Sk
inp
rim
ary
test
;C
on
jun
ctiv
alir
rita
tio
n(d
raiz
e)te
st;
Sk
inse
nsi
-ti
zati
on
test
;In
trat
rach
eal
inst
illa
tio
n
Sk
inp
rim
ary
and
eye
irri
tati
on
test
(rab
bit
s);
Sk
inse
nsi
-ti
zati
on
test
(gu
inea
pig
s);
per
ora
lad
min
istr
atio
nte
stan
d
NA
Intr
atra
chea
lin
stil
lati
on
and
per
ora
lad
min
istr
atio
nro
ute
s
Qu
ite
low
-acu
tep
ero
ral
tox
-ic
ity
was
fou
nd
(42
00
0m
g/k
gb
od
yw
eig
ht)
Co
rnea
As-
gro
wn
SW
CN
Hs
fou
nd
no
n-
irri
tan
tan
dn
on
-der
mal
sen
siti
zer
No
corn
eal
inju
ryw
aso
bse
rved
Miy
awak
iet
al.
(20
08
)
(co
nti
nu
ed)
DOI: 10.3109/17435390.2016.1153165 Please check 9
Dow
nloa
ded
by [
Tex
as A
&M
Uni
vers
ity L
ibra
ries
] at
06:
58 3
1 M
arch
201
6
Tab
le1
.C
on
tin
ued
Nan
om
ater
ials
Ex
vivo
stu
dy
Invi
vost
ud
yT
her
apeu
tic
del
iver
edR
ou
tes
of
adm
inis
trat
ion
Tox
icit
yT
arget
site
Ou
tco
me
Ref
test
;P
ero
ral
adm
inis
trat
ion
test
intr
atra
chea
lin
stil
lati
on
(rat
s)
iney
eir
rita
-ti
on
test
Cd
Se/
Zn
Sco
re/
shel
lQ
Ds
Fre
shly
der
ived
bov
ine
corn
ea;
bov
ine
cor-
nea
lfi
bro
bla
st(B
CF
)an
db
ov
ine
skin
fib
rob
last
(BS
F)
C5
7B
L/6
Jm
ice
No
tu
sed
Intr
astr
om
alin
ject
ion
Acc
um
ula
tio
no
fQ
Ds
into
the
corn
eal
stro
-m
alce
lls
Co
rnea
Th
eQ
Ds
get
accu
mu
late
wit
hin
the
corn
eas
for
lon
ger
du
r-at
ion
(26
d)
Ku
oet
al.
(20
11
)
Hu
man
reti
nal
pig
men
tep
i-th
eliu
m(R
PE
)
Rab
bit
sN
ot
use
dIn
trav
itre
alin
ject
ion
Lit
tle
chan
ge
iney
ebal
lap
pea
ran
ce,
IOP
,an
dey
esig
ht
Vit
rou
sG
Od
idn
ot
cau
sean
ysi
gn
ifi-
can
tto
xic
ity
toth
ece
llg
row
than
dp
roli
fera
tio
n.
Intr
avit
real
inje
ctio
no
fG
Oin
tora
b-
bit
s’ey
esd
idn
ot
lead
tom
uch
chan
ge
inth
eey
ebal
lap
pea
ran
ce,
IOP
,el
ectr
o-
reti
no
gra
m,
and
his
to-
log
ical
exam
inat
ion
Yan
etal
.(2
012)
Co
llo
idal
go
ldn
ano
rod
sE
xv
ivo
on
po
r-ci
ne
eyes
Po
rcin
e–
Las
erp
uls
ead
min
istr
atio
nT
her
mal
dam
age
con
firm
edC
apsu
les
Th
erm
ald
amag
ew
ith
in5
0–
70mm
inra
dia
ld
ista
nce
Rat
toet
al.
(20
09
)
Hea
t-se
nsi
tive
or
hea
t-an
dp
H-
sen
siti
ve
lip
oso
mes
HU
VE
Can
dA
RP
ER
abb
its
Cal
cein
Intr
avit
real
inje
ctio
ns
Did
no
tsh
ow
san
yto
xic
ity
Ret
ina
Th
ep
H-
and
hea
t-se
nsi
tive
lip
oso
me
show
edti
me-
and
site
-sp
e-ci
fic
dru
gd
eliv
ery
Laj
un
enet
al.
(20
15
)
(co
nti
nu
ed)
10 N.K. Mehra et al. Nanotoxicology, Early Online: 1–25
Dow
nloa
ded
by [
Tex
as A
&M
Uni
vers
ity L
ibra
ries
] at
06:
58 3
1 M
arch
201
6
Nan
om
ater
ials
Ex
vivo
stu
dy
Invi
vost
ud
yT
her
apeu
tic
del
iver
edR
ou
tes
of
adm
inis
trat
ion
Tox
icit
yT
arget
site
Ou
tco
me
Ref
Su
bm
icro
n-s
ized
lip
oso
mes
(ssL
ips)
Neu
ron
alp
recu
r-so
rce
llli
ne
(RG
C-5
)
Alb
ino
dd
Ym
ice
Ed
arav
on
eIn
trav
itre
alad
min
istr
atio
nR
etin
ald
amag
eR
etin
aE
dar
avo
ne-
load
edli
po
-so
mes
red
uce
dox
i-d
ativ
est
ress
-in
du
ced
ret-
inal
dam
age
mo
rest
ron
gly
rath
erth
anfr
eed
rug
su
po
nin
trav
i-tr
eal
inje
ctio
n
Hir
on
aka
etal
.(2
01
1)
Ch
ito
san
–li
po
som
esN
ewZ
eala
nd
Alb
ino
Rab
bit
s
Dic
lofe
nac
sod
ium
Low
erco
nju
nc-
tival
sac
No
irri
tati
on
and
tox
icC
orn
eaL
CH
-co
ated
lip
oso
mes
show
edsi
g-
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Carbon nanomaterials
Currently, carbon-based nanomaterials (CNMs) have attractedattention in ocular therapeutics delivery and targeting. For CNMssuch as carbon nanohorns (CNHs), poly hydroxy fullerenes(PHFs), carbon nanorods (CNRs), carbon nanodots (CNDs),CNTs, and graphenes, their inherent toxicity due to the presenceof metallic and amorphous impurities restricts their application inocular delivery. On the other hand, the unique physicochemicalproperties and scalability have rendered CNTs as ideal candidatesfor targeted drug delivery, including ophthalmic administration.The CNTs are available in four categories depending on thenumber of walls, i.e., single- (SWCNTs), double- (DWCNTs),triple- (TWCNTs), and multi-walled CNTs (MWCNTs) (Lacerdaet al., 2012; Mehra & Jain, 2015a–c; Mehra & Palakurthi, 2015;Mehra et al., 2014a,b, 2015a; Sharma et al., 2015).
As we know, CNTs are present in some cosmetic products,such as the eye liner kohl (Kajal; Surma), used in Indian culture.Kohl obtained from various minerals has been used as an eyecosmetic since ancient times. It is believed by some that the kohlprotects eyes and provides strength to the infants’ eyes. Literaturesuggests that kohl (Kajal) has been used for the prevention andtreatment of eye diseases such as cataract, conjunctivitis andblepharitis (Mehra et al., 2015a–c). The increased production andbiomedical applications of new multifunctional CNTs haveincreased the exposure to the environment and public. Thus, thesafety, efficacy, and toxicity issues are crucial to ensure the safeuse of CNTs and to address the major concerns of non-biodegradability, high aspect ratio, and similarity with asbestosand vitreous fibers (Boczkowski & Lanone, 2012; Kayat et al.,2011; Jain et al., 2014,2015). Due to their needle-like tubularstructure, multifunctional CNTs may be useful in ophthalmicdelivery. Du et al (2013) explored the relationship betweenphysicochemical properties and production of CNTs, along withthe potential risks to human health with exposure to thesenanomaterials.
Two different kinds of multi-walled CNTs (MWCNT 1: 5–8mm in length with 3–8 nm inside diameter and outside diameterof 140 ± 30 nm and MWCNT 2: 1–10 mm in length with 2–6 nminside diameter and outside diameter of 10–15 nm) have beenused in an eye irritation test in the rabbit (Kishore et al., 2009).Eye irritation is summarized as a ‘‘maximum average score’’,which an average value is scored based on corneal injury forindividual animals at the time of scoring. The MWCNTs weredispersed in distilled water with vortexing and the average particlesize (nm) and polydispersity index (0 to 1 with 0¼monodisperseand 1¼ polydisperse) for MWCNT 1 was 901 nm and 1, and forMWCNT was 554 nm and 1.000 and 1.00, respectively The meantissue viability of C1 (the size of MWCNT 1 was 166 nm), C2 (thesize of MWCNT 2 was 100 nm was450%, considered to be a non-irritant in SkinEthicTM reconstructed human epidermis skincorrosion test. A total of about 18 mg (an equivalent weight to0.1 mL) of the test substance (MWCNT 1 and MWCNT 2) wasapplied into the conjunctival sac of the left eye after gently pullingthe lower lid away from the eyeball, while the right eye served asthe control. The conjunctiva, iris and cornea of both eyes wereevaluated and scored according to the Draize method up to 72 h.Both sizes of MWCNTs were minimally irritating in the in vivoocular test (Kishore et al., 2009).
Ema et al. evaluated the eye irritation of two different kinds ofMWCNTs (Nikkiso-MWCNTs and MWCNT-7) and single-walled CNTs (SWCNTs), Nikkiso-SWCNTs and super-growthSWCNTs, using an eye irritation test in rabbits. After instillationof either the MWCNTs or SWCNTs into the conjunctival sac,there were no signs of corneal opacity, abnormality of the iris orchemosis (swelling of conjunctiva) in accordance to the
Organization for Economic Co-operation and DevelopmentGuideline 405 ‘‘Acute Eye Irritation/Corrosion’’ (OECD, 2002).Hyperanemia of the conjunctival blood vessels was observed 24 hfollowing instillation of the Nikkiso-MWCNTs, but this resolvedwithin 48 h. There also was no evidence of corneal epitheliumdamage. Overall, the SWCNTs and MWCNTs in this studyappeared to exhibit minimal eye irritation (Ema et al., 2011).
The ocular biocompatibility of plasma-modified MWCNTs(pristine, hydroxyl, and carboxyl modified MWCNTs) withhuman retinal pigment epithelium (RPE) cells was reported forthe first time by Yan et al. (2013). The reactive oxygen speciesgeneration was visible but not severe; however, MWCNTs-carboxyl exposure promoted a low oxidative stress level. Theplasma- and carboxyl-modified MWCNTs showed better ocularbiocompatibility (Lu et al., 2013). Although CNTs were widelyinvestigated for drug delivery and targeting, few studies havereported their toxicological properties (Jain et al., 2007).
Carbon nanohorns
CNHs represent a new type of CNMs and comprise of singlegraphene tubes (2–5 nm in diameter and 40–50 nm in tubulelength) with a conically closed tip. The CNHs (approximately2000 horns) form spherical aggregates with a diameter of 80–120 nm, which are reminiscent of dahlia flowers. They also formbuds and seeds and can target and accumulate within tumorsbecause of an enhanced permeability and retention (EPR) effect(Guerra et al., 2012). Additionally, CNHs do not contain metallicimpurities, a common cause for any toxicity of carbonnanomaterials.
In vitro and in vivo toxicity studies of as-grown SWCNHs(tubular nanocarbons having no metallic impurity) were per-formed by Miyawaki et al. (2008). The authors reported that theas-grown SWCNHs did not show any signs of conjunctivalirritation (Draize score¼ 0) in rabbits. Figure 3 shows no cornealinjury in the rabbit eye after an eye irritation test.
The international standard assay for acute ocular toxicity is therabbit in vivo Draize eye test (Draize et al., 1994), which wasdeveloped by the Food and Drug Administration. The procedureinvolves applying 100ml (100 mg if solid) of the test substanceonto the cornea and the conjunctival sac of one eye of the
Figure 3.Photo images of rabbit eye after eye irritation test: (a) untreated;(b) 1 h after SWCNHs dose in conjunctival sac without eye washing(reproduced with copyright permission from Miyawaki et al., 2008,American Chemical Society).
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conscious rabbit. Rabbits are observed up to 21 d at regularintervals for signs of irritation including redness, swelling,cloudiness, edema, hemorrhage, discharge, and blindness(Huhtala et al., 2008).
Graphenes
Graphenes are two-dimensional, monoatomic thick buildingblocks of carbon atoms packed into a honeycomb lattice. Theyshow great potential in drug delivery due to excellent biocom-patibility and aqueous dispersibility. Graphene oxide has highspecific surface area and large �-conjugated aromatic structure.Thus, various biomolecules such as doxorubicin, camptothecin,and paclitaxel can be loaded through �–� stacking interactions(Angelopoulou et al., 2015; Mao et al., 2013; Wang et al., 2011;Yang et al., 2015). However, pure graphene nanosheets areunsuitable for drug delivery and targeting due to their hydropho-bic nature and tendency to form agglomerates (Liu et al., 2015).
Up to now, reports on ocular biocompatibility of graphene andits derivatives are sparse. The toxicity and intraocular biocom-patibility of graphene oxide (GO) on human RPE cells werestudied by measuring cell viability and membrane integrity (Yanet al., 2012). The lactate dehydrogenase (LDH) assay was used tomeasure membrane integrity because LDH is a cytosolic enzymethat is released into the culture medium upon cell membranedamage (Hillegass et al., 2010). The RPE cells treated with GOsolutions displayed higher than 60% cell viability and less than 8%LDH release. An insignificant number of cells (1.5%) showedapoptosis. The authors also found that the intravitreal injection ofGO into the eyes of rabbits did not promote any changes in eyeballappearance, eyesight, intraocular pressure (IOP), electroretino-gram measurements, and histopathology (Figures 4 and 5). Theirfindings clearly revealed the potential of GO for clinicaldiagnosis, imaging and drug delivery applications in ophthalmol-ogy (Yan et al., 2012).
Figure 5. Digital camera of the rabbit’s eyeball from (a) the control eye, and (b) the 0.3 mg of GO-injected eye after injection for 49 d. Histologicalappearance of (c) the control retina and (d) the 0.3 mg of GO-injected retina after injection for 49 d (reproduced with copyright permission from Yanet al. 2012, American Chemical Society).
Figure 4. Digital photos of the experimental rabbit (top) and slit-lampfundus photo of the control eye (bottom). Slit-lamp fundus photos of (b)intravitreally injected eyes after the injection of 0.1 mg of GO, (c) 0.2 mg of GO, and (d) 0.3 mg of GO for 2 (top) and 49 d (bottom) (reproduced withcopyright permission from Yan et al., 2012, American Chemical Society).
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More recent reports by Chen et al. (2015) studied thenanotoxicity of GO during zebrafish embryogenesis and foundthat GO damaged the mitochondria and induced developmentalmalformation of the eyes. An alternative graphene derivative toGO that has been considered for ocular delivery is hydroxylatedgraphene (G–OH). The genotoxicity and ocular biocompatibilityof G–OH prepared by ball milling was reported by Lin et al.(2015). Genotoxicity and in vivo ocular biocompatibility studiesare essential for large-scale up and commercialization of G–OH.The possible genotoxicity (using the comet assay to measuredeoxyribonucleic acid strand breaks) was induced by G–OHat4100 mg/mL concentrations. Intravitreal injection of G–OH inrabbits gradually decreased genotoxicity after 4 weeks devoid ofdamage to cell morphology, structure, and most parts of the eyes.The authors also reported that the G–OH caused some changes ineyesight-related functions (IOP, ERG, and retinal structureschanges) (Lin et al., 2015). Thus, suitable eye protection maybe recommended for people who are dealing with graphene-basedmaterials frequently in the laboratory.
Fullerenes
Fullerenes are the third stable allotrope of carbon (in addition tographite and diamond) and were discovered in 1985. They are
spherical carbon cages with sp2 hybridized molecules and consistof 60 carbon atoms. Polyhydroxy-fullerenes (PHF) are watersoluble, negatively charged, biocompatible, functionalized fuller-enes molecules. The potent antioxidant nature of PHF hasattracted great attention in pharmaceutical applications.However, only a few studies have reported on the toxicity andsafety of highly purified fullerenes based on the screening of eyeand skin damage.
Our research group has determined the toxicity of carboxy-and hydroxy-terminal PHF in rabbit corneal epithelial (RCE) cellsand porcine retinal arterioles in vitro. The RCE cells were treatedwith different concentrations of PHF (carboxy terminal PHF; F1;hydroxy terminal PHF; F2) for 24 h at 37 �C and the cell viabilitywas assessed by the methyl thiazoletetrazolium assay andpresented in Figure 6. The IC50 values of the F1 and F2 werecalculated to be 26.8 and 27.5 mM, respectively. To study thedirect and acute impact of nanomaterials on vasomotor functionof retinal arterioles isolated from pigs, the concentration-dependent vasodilation to known endothelium-dependent nitricoxide-mediated vasodilator bradykinin (1 pM–10 nM) was exam-ined in the absence or presence of F1 and F2 for up to 2 h. Asshown in Figure 7, bradykinin at 0.1 nM produced significantdilation of the isolated retinal arteriole (i.e., the diameter wasincreased from the resting state of 45–57 mm; Figure 7A). At a
Figure 6. Percent cell viability of rabbitcorneal epithelial cells after treatment withcarboxy terminal PHF-F1; hydroxy terminalPHF-F2 nanomaterials at 0.01–10 mM. Valuesrepresented as mean ± SD.
Figure 7. (A) Retinal arteriolar images observed on the video monitor for internal diameter recordings. The diameter was recorded in the absence ofagonists (resting tone) and in the presence of bradykinin. (B) Retinal arterioles dilated to bradykinin. The vasodilations were reduced after treating thevessels with 1 mM carboxy terminal PHF-F1nanomaterials for 2 h. The dilations were not affected by 0.3 mM F1 (n¼ 4 for each group, ANOVA*p50.05).
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higher concentration (10 nM) of bradykinin, the vessel furtherdilated to 83 mm. Interestingly, when vessels were treated with F1at 1mM intraluminally for 2 h, the concentration-dependentdilation to bradykinin was significantly impaired (Figure 7A).The low concentration of F1 (0.3 mM) did not alter the dilation tobradykinin (Figure 7B). The lack of toxicity was also observedwith F2 nanomaterials at 0.3 mM (data not shown). These pilotdata suggest that F1 exerts an adverse effect possibly on theendothelial cells and compromises the vasodilator function ofretinal arterioles.
Experimental studies have also examined the effect offullerene soot and highly purified fullerenes on the eye. Huczkoet al. (2001) reported no eye irritation in guinea pigs withfullerene soot in the draize eye irritation test. In addition,Aoshima et al. (2009) performed the primary eye-irritation test inrabbits and observed conjunctival redness and corneal epitheliumdefects following fullerenes application (presumed to be due toinsoluble fullerene powder with an average particle size of 100–300mm), but the symptoms disappeared within 48 h.
Metallic nanoparticles
Metallic nanoparticles including nanogold, nanosilver, quantumdots, and metallic oxides have been used in ocular delivery. Up tonow, limited studies have been performed for in vivo corneal genetherapy using gold nanoparticles (GNPs). It is known that bothacquired and genetic factors may affect the corneal transparencyand may lead to the loss of vision (Sharma et al., 2011). Readersmay refer to ‘‘silver nano-a trove for retinal therapies’’ for a moredetailed account on retinal therapies (Kalishwaralal et al., 2010).
The gene transfer potential and toxicity of 2-kDa polyethyle-neimine (PEI) conjugated gold nanoparticles (PEI2-GNPs) werestudied in the human cornea in vitro and the rabbit cornea in vivo(Sharma et al., 2011). Exposure of primary human cornealfibroblasts isolated from donor corneas to PEI2-GNPs mixed withDNA plasmids allowed for effective gene delivery withoutdamaging the cells. The topical route of application, as performedin the in vivo study with PEI2-GNPs, is the most convenient routefor delivering therapeutics into eye because of the absence ofpharmacokinetic challenges. The slit-lamp biomicroscopy clearlyrevealed the lack of edema, redness, and cellular infiltration in thePEI2-GNP-treated rabbit corneas, suggesting low toxicity withcorneal PEI2-GNP therapy (Figure 8). Transmission electronmicroscopy studies confirmed the internalization of PEI2-GNPs
in corneal cells in vivo via an endocytic mechanism after topicalapplication with gradual clearance over time (Figure 9). Thefindings suggest that GNPs did not harm corneal cells and theyslowly cleared from the rabbit cornea. Low toxicity, rapid uptake,and slow clearance of the PEI2-GNPs may open new doors in thecoming days to the ophthalmologist and researchers.
The internalization, apoptosis, oxidative stress, glial, andmicroglial activities of silver and GNPs on C3H wild-type miceretinal tissue were studied (Soderstjerna et al., 2014). The NP-related neuronal toxicity was evident by the higher number ofapoptotic cells, oxidative stress, and increased glial staining andmicroglial activation (hallmark of neural tissue insult). Lowconcentrations of Ag- (20 nm size) and AuNPs (80 nm size)showed adverse effects on the retina, using an organotypic retinaculture model.
Quantum dots
QDs are semiconductor nanocrystals of groups III–IV and II–VIin the range of 1–10 nm in diameter (Bajwa et al., 2015a,b;Volkov, 2015). Along with their small size and variable shape,they exhibit unique optical properties such as tunable emission,strong brightness, high photostability, and high resistance tophotobleaching, which has attracted attention for investigations indrug delivery, targeting, and imaging. A few studies have beenreported on the safety, routes of administration, and toxicity. In arecent study, the impact of the common CdSe/ZnS core/shell QDson the cornea was evaluated in bovine eyes in vitro and mice eyesin vivo (Kuo et al., 2011). The exposure to QDs at a concentrationof 20 nM for 48 h reduced the viability (50%) of bovine cornealfibroblasts (Figure 10). Twenty-six days after intrastromal injec-tion in the mice cornea, these QDs penetrated and were retainedwithin the corneal stroma (Figure 11). Because corneal stromacells play an important role in the maintenance of the health andtransparency of the cornea, these findings suggest potentialtoxicity with prolonged accumulation of QDs. Thus, furtherresearch is required to examine the exposure and possible risk ofQDs for ocular applications.
Dendrimers
Dendrimers (from the Greek word ‘‘Dendron’’ for tree) arehyperbranched synthetic macromolecules with monodisperse(uniform) repeating units and a globular three-dimensional
Figure 8. Representative clinical eye examination images acquired with a slit-lamp microscope in PEI2-GNP-treated and untreated (control) rabbitcorneas at 12 h and 7 d post-application. No opacity, redness, or inflammation was seen. PEI2-GNP-treated corneas show mild purple coloration at 12 h,confirming GNP uptake, and clearing of coloration detected on day 7 suggests that there was no GNP accumulation in the cornea (reproduced withcopyright permission from Sharma et al., 2011, Elsevier Pvt Ltd).
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morphology. They have been used as one of the promisingnanomaterials in ocular drug delivery. While the cationic natureof amine-terminated dendrimers enhances the epithelial mem-brane permeability, strong interactions with the negatively
charged membrane may result in toxicity (necrosis and apoptosis).Thus, surface engineering is needed to alleviate toxicity of thecationic dendrimers (Jain et al., 2015; Mehra et al., 2015a–c;Mishra & Jain, 2014; Yellepeddi et al., 2013).
Figure 10. Cell viability of QD-corneal fibroblast using MTT assays. *significant compared with the control (bovine cornea fibroblast incubatedwithout QDs) (reproduced with copyright permission from Kuo et al., 2011 American Chemical Society).
Figure 9. Representative TEM images of PEI2-GNP-treated rabbit corneas demonstrating the presence and intracellular trafficking of GNPs in (A)keratocytes and (B) extracellular matrix. GNPs can be seen in the endosomes near the cell surface indicating their uptake by endocytosis. Rupturedendosome represents the release of GNPs into the cytoplasm (reproduced with copyright permission from Sharma et al., 2011, Elsevier Pvt Ltd).
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Experimental in vivo evidence has been provided to supportthe potential use of dendrimers as drug carriers in the eye. Acommon type for drug delivery is poly(amidoamine) (PAMAM)dendrimers. The bioadhesive properties and strong interaction ofPAMAM dendrimers with the corneal surface likely contributedto the increased bioavailability of pilocarpine (an anti-glaucomamedication) and tropicamide (anticholinergic drug used to dilatethe pupil) following ocular instillation of the PAMAM–drugsolutions in rabbits (Vandamme & Brobeck, 2005).The PAMAMdendrimers appear to be safe for topical application because theydid not cause ocular irritation. Another type of dendrimer-containing phosphorous was synthesized and tested for its abilityto improve ocular delivery of carteolol (an ocular anti-hyperten-sive drug used in treating glaucoma) in vivo in rabbits (Spataroet al., 2010). Although solubility of the carteolol–dendrimersmixture was low, this formulation did not cause irritation up to 8 hpost-instillation on the cornea and increased the amount ofcarteolol delivered to the aqueous humor. In a recent study,Mishra and Jain (2014) developed a topically effective acetazo-lamide (ACZ)-loaded poly(propylene imine) (PPI) dendrimers(ACZ/PPI) for glaucoma. The ACZ/PPI dendrimers enhancedocular residence time and showed efficacy in lowering IOP. Theocular irritation toxicity of ACZ/PPI dendrimers from themodified Draize test revealed that the dendrimer formulationswere safe for ocular delivery.
In the United States, atrophic (dry) age-related maculardegeneration (AMD) and retinitis pigmentosa (RP) are leadingcauses of blindness and no effective treatments are currentlyavailable for these chronic retinal diseases. Retinal neuroinflam-mation triggered by activated microglia appears to play a pivotalrole in the retinal pigment epithelial and photoreceptor cell loss inRP and AMD. In a recent study, hydroxyl-terminated PAMAMdendrimers were found to selectively localize within activatedmicroglia following intravitreal injection in the Royal College ofSurgeons (RCS) rat retinal degeneration model (Lezzi et al.,2012). The dendrimers were then conjugated to fluocinoloneacetonide and tested for their efficacy in drug delivery. In the RCSrats, there was a greater reduction in neuroinflammation andretinal degeneration one month after intravitreal administration ofthe dendrimers-steroid formulation than the free steroid alone.These findings along with the ability of only the dendrimers-steroid formulation to preserve neural retina function (electro-retinogram assessment) over 9 weeks suggest that these dendri-mers are safe for intravitreal delivery.
Glaucoma is a neurodegenerative disease with increased IOP,oxidative stress, and impaired ocular blood flow and it is a major
cause of irreversible blindness and visual impairment. Accordingto the World Health Organization, approximately 67 millionpeople are affected worldwide and the projected figure will reach80 million by the year 2020. A single therapeutic agent cannotoften control the IOP because of the multifactorial nature ofglaucoma and due to short duration of action. A combination oftwo or more anti-glaucoma therapeutic agents is a better way tocontrol and reduce IOP as well as to improve patient compliance.Up to now, various combinations of therapeutic agents have beendeveloped and are available in the market including latanoprost/timolol maleate, dorzolamide/timolol maleate and brimonidinetartrate/timolol maleate (Desai et al., 2010; Holden et al., 2012;Yang et al., 2012). Recently, Yang et al. (2012) co-deliveredtimolol maleate and brimonidine using a novel hybrid PAMAMdendrimers hydrogel/poly (lactic-co-glycolic acid) (PLGA) nano-particle platform (HDNP) as an eye drop formulation in normo-tensive adult Dutch-belted rabbits. After one-time topicalapplication, the HDNP formulation led to a sustained andeffective reduction in IOP for 4 d. This treatment also signifi-cantly increased the absorption of both anti-glaucoma agents inthe aqueous humor and cornea as well as absorption of timololmaleate in the conjunctiva of the eye for 1 week (Figure 12)without promoting inflammation or structural changes in the eye.Collectively, these results support the ability of a newly developedHDNP to enhance drug bioavailability and sustain IOP reductionfor several days with potential clinical application for improvinglong-term patient compliance and reducing dosing frequency aswell the enormous costs in ocular therapy.
Nanowafers
Dry eye disorder is one of the major public health issues and acommon eye disease, affecting millions of people worldwide.About 30% of patients have reported symptoms of mild or chronicdry eye disease. It is characterized by blurred vision, eyeirritation, light sensitivity, and ocular surface epithelial disease.Injuries on the surface of the eye due to dry eye disease disruptcorneal angiogenic privilege and trigger corneal neovasculariza-tion, which can lead to loss of vision (Azar, 2006; Coursey et al.,2015; Yuan et al., 2015). Dry eye is a multifactorial disease andactivates an innate immune response in the conjunctiva andcornea (Coursey et al., 2015). Currently, artificial tears and anti-inflammatory eye drops are being used for the treatment of dryeye disease and the eye drops are administered several times a dayto reach therapeutic efficacy. Because drug loaded contact lensescould not release the drug for an extended period of time,
Figure 11. (a) In vivo external photographs of mice corneas following an intrastromal injection of PBS (right eye) and QDs (left eye). (b) Themagnified image of the left eye. (c) Two-photon imaging of mice corneas 26 d following intrastromal QD injection. The arrow indicates theintracytoplasmic-localized QDs (reproduced with copyright permission from Kuo et al., 2011, American Chemical Society).
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nanowafers were developed. Nanowafers are readily applied bythe fingertip to the eye like a contact lens. This technology wasdeveloped by Acharya et al. (2015) in the Department ofOphthalmology at the Baylor College of Medicine. The nanowaferis a small/tiny transparent circular disc that contains arrays ofdrug-loaded nanoreservoirs. It can be applied on to the ocularsurface using a fingertip and it withstands constant blinkingdevoid of being displaced. The nanowafers delivery system couldpotentially be used for glaucoma, eye injuries, chronic dry eye,infections, and other ocular inflammatory conditions (Courseyet al., 2015; Yuan et al., 2015). Nanowafers release the drug for alonger duration of time, and subsequently the device slowlydissolves as the drug is released. Nanowafers can be easily placedonto the ocular surface, which could deliver therapeutics moreconsistently for longer duration of time to the eye as well asreduce the frequent administration of dosage forms and providebetter efficacy and feasibility. The dexamethasone (Dex) loadednanowafers (Dex-NW) were developed to deliver drugs on theocular surface for a longer duration of time (Coursey et al., 2015).Dex is a glucocorticoid with potent anti-inflammatory propertiesand is well documented for its efficacy in treating ocularinflammation and dry eye-related corneal epithelial diseases.The Dex-NW contains carboxy methyl cellulose and a 500 mmsquare drug reservoir, which were filled with Dex. The device waseffective in downregulating expression of chemokines (CXCL-10and CCL-5) and inflammatory cytokines (TNF-a and IFN-g).
Acharya et al. (2015) selected four different polymers,poly(vinyl alcohol), polyvinyl pyrrolidone, (hydroxyl pro-pyl)methyl cellulose, and carboxylmethyl cellulose (CMC), fordesigning and fabricating Nanowafers via hydrogel templatestrategy. These polymers are hydrophilic, transparent, biocom-patible, and mucoadhesive and have film-forming properties,
which allow them to readily adhere onto a wet mucosal surfaceand conform to the curvature of the eye. These fabricatedNanowafers contained sunitinib, sorafenib, and axitinib, which arealready in clinical use as anti-angiogenic therapeutics to treat late-stage renal carcinoma (Yuan et al., 2015). In a murine ocular burnmodel, once-a-day administration of axitinib-loaded nanowaferswas more effective than twice-a-day therapy with axitinib eyedrops in treating corneal neovascularization (Yuan et al., 2015).The afitinib-nanowafers were also non-toxic and did not disruptthe wound healing of the cornea. These findings suggest thatnanowafers could provide a safe and effective drug deliverysystem at a lower dosing frequency for ocular therapy.
Hydro- and nano-gel
Hydrogel is a polymer network that is expanded throughout itswhole volume by water. Nanogels are hydrogels consisting of ananoscale hydrophilic polymer network, which is loaded with adrug and can be used for delivering both hydrophilic andhydrophobic drugs. Based on how polymer networks areformed, hydrogels can be classified into physical and chemicalgels. A wide variety of synthetic, semi-synthetic and naturalpolymers such as hyaluronic acid, PVA, chitosan, acrylatecopolymers, collagen, alginate, and poly(ethylene glycol areused for hydrogel formulations (Kirchhof et al., 2015; Kompellaet al., 2013).
Toxicity evaluation of biodegradable and thermosensitivepoly(ethylene glycol)–poly(epsilon-caprolactone)–poly(ethyleneglycol) hydrogel as an in situ sustained ophthalmic drug deliverysystem showed slight corneal endothelial damage (Yin et al.,2010). The ion-sensitive hydrogels were developed using gellangum, kappa-carrageenan, and alginates (natural polysaccharides)
Figure 12. Representative fundus cameraimages of rabbits instilled with HDNP andNP formulations. FluoSpheres wereentrapped in dendrimer hydrogel or PBS pH7.4 instead of drug-encapsulating PLGAnanoparticles. Images were taken at the endof days 1, 3, 5, and 7 of instillation offormulations. Left panel of each group is theregular fundus camera image of the eyes, andright panel is a fluorescence fundus cameraimage (reproduced with copyright permissionfrom Yang et al., 2012, American ChemicalSociety).
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for ophthalmic drug delivery systems. To assess the ocular safety,the ion-sensitive hydrogel was instilled into the eye of maleSprague–Dawley rates every 12 h for 3 months. No signs ofcorneal anterior surface damage, conjunctival injury, redness, orincreased tearing were observed by direct visual observation or byimmunohistochemical techniques (Figures 13 and 14)(Fernandez-Ferreiro et al., 2015).
Liposomes
Liposomes are biocompatible and biodegradable lipid assembliesand were evaluated for the first time in the 1980s with the mainobjective of enhancing corneal penetration of ocular therapeutics.Liposomes may vary in size, lipid composition, surface charge,method of preparation, and fluidity of bilayers. Liposomes havinga positive charge and nano-size range can lead to prolongedresidence time with improved therapeutic effects. The liposomescan encapsulate both hydrophilic and hydrophobic drugs and
release them through passive diffusion, vesicle fusion, and vesicledisruption (Kompella et al., 2013; Reimondez-Troitino et al.,2015). Liposomes may cause blurred vision and a possibility ofaggregation during storage or in vivo when colloidal stability ispoor. In addition, cationic surface charge of liposomes might beresponsible for inducing inflammation (Bochot & Fattal, 2012).Pattni et al. (2015) very recently reviewed ‘‘New Developments inLipsomal Drug Delivery’’ in Chemical Reviews Journal.
In 2007, positively charged multi-lamellar liposomes com-posed of egg phosphatidylcholine:cholesterol:stearylamine (7:7:1molar ratio) were prepared using the reverse-phase evaporationmethod and then loaded with the ophthalmic drug ACZ. Theliposomes showed ACZ percent encapsulation efficiency(48.27%). The authors reported that the study failed concerningthe effect of the liposomal formulation on improving ocularresidence time and ocular irritancy (Hathout et al., 2007).
Chitosan is a polysaccharide that is derived from crustaceanshells and is used in ocular drug and gene delivery. It is a natural
Figure 13. Macrophotography of the right eye of a Sprague–Dawley rat after the instillation of 7.5 mL of the ion-sensitive hydrogel containing Trypanblue (eeproduced with copyright permission from Fernandez-Ferreiro et al., 2015, Elsevier Pvt Ltd).
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cationic polymer with biocompatibility and mucoadhesive nature.Because of its cationic nature, chitosan nanoparticles can easilybind with the negatively charged cornea and maximize the cornealpenetration (Li et al., 2009; Prow, 2010). The low molecularweight chitosan (LCH, 8 kDa)-coated liposomes were preparedand assessed for in vitro and in vivo ocular delivery of diclofenacsodium (Li et al., 2009). From the ocular tolerance study, noirritation or toxicity was observed even after continual adminis-tration of the LCH-coated liposome in New Zealand albinorabbits for a total period of 7 d.
Poly-L-lysine (PLL) is a water-soluble cationic peptide and isused as a modifier for drugs. It enhances the cellular uptake ofdrugs by increasing the permeability of various compounds. PLLwas coated on the surface of submicronized (100 nm) liposomesand toxicity was determined in human corneal (RIKEN Cell Bank)and conjunctival (Wong–Kilbourne derivative of Chang conjunc-tiva, clone 1–5c-41, CCL-20.2) cell lines and interocular distribu-tion was assessed following eye drop administration in mice. Thefindings suggest that the PLL-modified liposomes are non-toxic incorneal or conjunctival cells and are effective for drug delivery tothe retina as eye drop formulations (Sasaki et al., 2013).
Hironaka et al. (2011) developed edaravone-loaded liposomesfor protection against oxidative stress-induced retinal damage.The retinal damage was induced by intravitreal injection of N-methyl-D-aspartate in Hank’s balanced salt solution (HBSS-Mesbuffer).The edaravone-loaded liposomes reduced oxidative stress-induced retinal damage more strongly than the free drug uponintravitreal injection. The results suggested that the edaravone-loaded liposomes remarkably improved the in vivo efficacy(Hironaka et al., 2011).
In another study, a liposomal formulation did not show anytoxic effects on human umbilical vein endothelial cells (HUVECs)and human retinal pigment epithelial (ARPE-19) cells. Theliposomes were developed employing heat-sensitive or heat- andpH-sensitive lipid composition with rod- or star -shapednanoparticles (Lajunen et al., 2015).
Micelles
Polymeric micelles are 10–100 nm self-assemblies of amphiphilicblock copolymers above critical micellar concentration (CMC)with a hydrophobic core surrounded by a hydrophilic shell.Depending on the solvent and amphiphilic molecule choice,different micellar structures are formed (standard, reverse, andunimolecular micelles). Amphiphilic block copolymers are easilytailored to prolong the micelles stability in eyefluids, inducebioadhesion, and modify drug release. The polymeric micelleshave been shown to improve drug permeation across the ocularepithelia with minimal or no irritation, leading to enhanced ocularbioavailability (Kaur & Kakkar, 2014; Kompella et al., 2013).
The itraconazole-incorporated amphiphilic block copolymer-based polymeric micelles (optimized micelles formulation) with asize of 79.99 nm, 91.32 ± 1.73% entrapment efficiency, and invitro percentage permeation (90.28 ± 0.31%) in an in situ oculargel was developed for the management of fungal keratitis (Jaiswalet al., 2015). The optimized itraconazole polymeric micellesloaded (optimized in situ gel) in an in situ ocular gel (0.5% w/vcarbopol 943 NF, 6.84 ± 0.34 pH, 47.3 ± 4.50 gelling time inseconds, and 82.22 ± 1.91% drug content) showed superior ex vivotranscorneal permeation as compared with Itral
�eye drops (Jawa
Pharmaceuticals, Gurgaon, India). No irritation was observedeven after repetitive use of the micelle formulation. Antifungalactivity was performed using the agar cup plate method withCandida albicans. The in situ gel showed a nearly 19 nm zone ofinhibition, which was higher than the zone of inhibition (about14 nm) with Itral
�eye drops.
Nanoemulsion
Nanoemulsions are composed of two immiscible phases (liquids),wherein one phase is dispersed as droplets in another phase andstabilized by surfactant (stabilizer). The nanoemulsion can beprepared using low viscosity grade surfactant, oil, and water. Thepresence of surfactant or in combination with co-surfactantsreduces the interfacial tension within nanoemulsion to facilitatedispersion, increases membrane permeability, and enhances druguptake. The non-toxic, non-irritant nature of surfactant, oil, andco-surfactants to the corneal surface and other ocular tissues/cellsis an important parameter (Daull et al., 2013; Gallarate et al.,2013; Lallemand et al., 2012; Kaur & Kakkar, 2014; Lee et al.,2015).
Difluprednate (6a, 9-difluoro-11b,17,21-trihydroxypregna-1,4-diene-3,20-dione 17-butyrate 21-acetate; DFBA) is a syn-thetic glucocorticoid used for the treatment of post-operativeinflammation and pain. DFBA is hydrolyzed into deacetylatedmetabolite 6a,9-difluoro-11b,17,21-trihydroxypregna-1,4-diene-3,20-dione 17-butyrate (DFB) in the aqueous humor afterinstillation into the eye. However, DFB shows similar activityas that of DFBA (Yamaguchi et al., 2005). DUREZOL
�(0.05%
difluprednate ophthalmic emulsion), a topical corticosteroid, isindicated for the treatment of inflammation, pain associated withocular surgery, and endogenous anterior uveitis. Safety andefficacy studies of DUREZOL
�with two or four times a day
application were conducted in 438 patients in 26 clinics in theUnited States. Based on the results, only 3% of patients showedclinically significant IOP rise in both DUREZOL
�groups
(�10 mm Hg and�21 mm Hg from baseline, respectively). Theauthors did not report any serious ocular adverse events and feweradverse events were found in difluprednate-treated groups ascompared with the placebo group (Korenfield et al., 2009).
Cyclosporine A (CsA) is a potent immunosuppressive agent. Itinhibits proliferation and action of T lymphocytes by blocking thetranscription of cytokines (Liang et al., 2012). CsA is useful forthe treatment of various ophthalmic diseases (dry eyes, uveitis inchildren and adolescents, peripheral ulcerative keratitis, andvernal keratoconjunctivitis) (Kapoor & Chauhan, 2008). Thesafety profile of three different CsA cationic emulsion formula-tions: (i) 0.05% CsA-CEm, (ii) commercial 0.05% CsA-anionicemulsion (CsA-AEm, Restasis
�), and (iii) 0.05% CsA-Oil solution
was tested in vitro with a dynamic corneal wound healing assayemploying HCE cells, and in vivo in a rabbit acute toxicity model(Liang et al., 2012). The modified Draize test scale was used forthe safety evaluation of conjunctival hyperemia, chemosis, andpurulent secretions upon instillation of the emulsions formulation.Upon repeated instillations, all three of the CsA emulsionformulations were well tolerated by the rabbit ocular surface.The acute toxicity of these emulsions was studied in a rabbit acuteirritation model. Phosphate-buffered saline (PBS) served as thenegative control, while 0.02% benzalkonium chloride (BAK) wasa positive control for toxicity. The CsA emulsion formulationsshowed a moderate irritation score (Draize test score ranging from2.6 to 4.5) at 75 min, which was higher than that obtained in thePBS-treated group (0.8) and lower than in the 0.02% BAK group.The 0.02% BAK caused the highest toxicity, chemosis withdamaged corneal epithelium, redness, and inflammatory cellinfiltration. However, CsA in anionic emulsion (CsA-A Em;Restasis
�; Allergan) and CsA in oil (CsA-Oil; medium-chain
triglycerides, Mygliol�
812; IMCD, Saint-Denis, France) inducedmoderate infiltrations of inflammatory cells near conjunctiva-associated lymphoid tissues structures (Liang et al., 2012).
Restasis is a microemulsion of cyclosporine (an immunosup-presive agent) and is used in the treatment of chronic dry eye,which may be caused by inflammation. The most common ocular
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side effects of Restasis, including ocular burning (17%), conjunc-tival hypermia, epiphora, pruritus, stinging and visual disturb-ances, eye pain, and foreign body sensation, have been reported in1–5% of patients (http://www.drugs.com/restasis).
A novel pH triggered nanoemulsified in situ ophthalmic gel offluconazole (NE-ISG) was designed, developed, and tested via exvivo application to the goat eye for transcorneal permeation andcorneal toxicity. These studies revealed that NE-ISG causedgreater permeation of fluconazole in the cornea than didcommercial eye drops of the antifungal agent without showingany visual signs of ocular tissue damage (Pathak et al., 2013). Acomplete list of various ophthalmic products available in themarket is shown in Table 2.
Conclusion
Currently, several types of nanomaterials are available fortherapeutic delivery and targeting to the eye in the treatment ofvarious eye-associated diseases. Surfactants are generally used toimprove the dispersibility and to reduce the size of the emulsionin both ocular nanoemulsions and nanosuspension formulations.The concentration of surfactant used has to be carefully evaluatedas higher surfactant concentrations because it may cause irritationand corneal damage. While ionic surfactants are toxic, non-ionicsurfactants like polaxamers, polysorbabtes, polyethylene glycol,and tyloxapol may be used at pre-assessed concentrations withoutsignificant toxicity. For example, polysorbate 80 is the mostwidely used in many ocular emulsion preparations and isconsidered safe.
Restasis� is a lipid emulsion containing of 0.05% CsA. Now-a-days contact lenses (Ocusert
�and contact lenses) have been
most widely used and studied owing to their higher degree ofcomfort and biocompatibility. As we know, drawbacks with someof the existing nanomaterials, such as nanoparticles (low loadingefficiency), dendrimers (toxicity due to peripheral functionalgroups), liposomes (stability issue) and carbon nanomaterials(accumulation in lungs), need to be resolved.
The selection of a suitable animal model in assessment ofocular toxicity of the nanomaterials and the product is importantfor the development of safe and effective ocular formulations. Therabbit eye is more sensitive and susceptible to irritant nanomater-ials than the human eye and is considered to be a suitable model.Toxicity of the nanomaterials-based ophthalmic formulation canbe best evaluated by corneal opacity, eye irritation (Draize eyetest), hypersensitivity, LVET, and ocular and non-ocular organo-typic methods. The corneal opacity test is generally performed onthe cornea of rabbits and it is most widely used in the assessmentof corneal toxicity. The LVET is a refinement of the Draize eyetest wherein a tenth of the amount (10 mL or 10 mg) of the testsubstance used in the Draize test is applied to the cornea (notconjunctival sac) of the right eye of the animal with no forcedeyelid closure employed. The pathological changes are observedin the cornea, conjunctiva and iris-ciliary body. While the Draizetest is often considered for its over-prediction of the humanresponse, LVET is considered more accurate (Roggeband et al.,2000). However, LVET is not adopted by any regulatory agencyas an alternate test. The reluctance to adopt the LVET may be dueto the fact that it does not offer the element of ‘‘exaggeration’’present in the Draize test that helps to assure public safety.Recently, legislation to reduce the animal testing methods and topromote alternative methods (deceased animal tissues, 2D and 3Dcell culture models) has been introduced in many countries. Acomprehensive review on current techniques for ocular toxicitywas presented by Wilson et al. (2015).
In conclusion, as novel nanomaterials are being developed,thorough physicochemical characterization followed by a batteryT
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of in vitro and in vivo ocular toxicity evaluation tests are neededfor a comprehensive understanding of the interactions ofnanoparticles at the molecular, cellular, and tissue levels. Theseinteractions would determine the biocompatibility, stability andbiological performance and side effects of the nanomaterials.Such a knowledge base will in turn allow in silico hazardassessment of the nanomaterials and guide the development ofbenign, next-generation nanomaterials.
Disclosure statement
The authors report that they have no conflicts of interest. Thecontent is solely the responsibility of the authors and does notrepresent the official views of the Food and Drug Administration.
Funding information
This work was supported, in part, by a grant from the Food andDrug Administration, Center for Drug Evaluation Research(U01FD05184) to S. P.
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