Cationic Gelatin Nanoparticles for Drug Delivery to the Ocular Surface: In Vitro and In Vivo ...

7/23/2019 Cationic Gelatin Nanoparticles for Drug Delivery to the Ocular Surface: In Vitro and In Vivo Evaluation

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Hindawi Publishing CorporationJournal o NanomaterialsVolume , Article ID ,pageshttp://dx.doi.org/.//

Research ArticleCationic Gelatin Nanoparticles for Drug Delivery tothe Ocular Surface: In Vitroand In VivoEvaluation

Ching-Li Tseng,1 Ko-Hua Chen,2,3,4Wen-Yu Su,2,5,6Yen-Hsien Lee,2,7

Chi-Chang Wu,1 and Fen-Huei Lin2,5

Graduate Institute of Biomedical Materials and issue Engineering, College of Oral Medicine, aipei Medical University, No. ,Wu-Hsing Street, aipei City , aiwan

Division of Medical Engineering Research, National Health Research Institutes, No. , Keyan Road, Zhunan own,

Miaoli County , aiwan Department of Ophthalmology, aipei Veterans General Hospital, No. , Section , Shipai Road, Beitou District,

aipei City , aiwan National Yang-Ming University, No. , Section , Linong Street, aipei City , aiwan Institute of Biomedical Engineering, National aiwan University, No. , Section , Ren-ai Road, aipei City , aiwanInstitute of Biomedical Engineering and Material Science, Central aiwan University of Science and echnology, No. , Buzih Road,

aichung City , aiwanGraduate Institute of Medical Science, College of Medicine, aipei Medical University, No. , Wu-Hsing Street,

aipei City , aiwan

Correspondence should be addressed to Fen-Huei Lin; [email protected]

Received August ; Revised November ; Accepted November

Academic Editor: Anchal Srivastava

Copyright Ching-Li seng et al. Tis is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

o develop an effective ocular drug delivery carrier, we prepared two different charged gelatin nanoparticles (GPs) and evaluatedparticle size, surace charge, and morphology. Tein vitrobiocompatibility o GPs was assessed using human corneal epithelium(HCE) cells andin vivosaety by administering them as eye drops to New Zealand rabbits. Te GPs prepared using type A gelatinwere positively charged (GP(+), + mV; size,180.6 45.7 nm). Water-soluble tetrazolium salt (WS)- assay showed that bothGPs were nontoxic to HCE cells. Te uorescence intensity o HCE cells cultured with cationic GPs conjugated with a uorescentdye was higher than that o the anionic GP-treated HCE cells. In vivoexamination showed no serious irritation to the rabbit eyes.Furthermore, corneal thickness and ocular pressure in the eyes o the treated rabbits were similar to those in the eyes o normalrabbits. Microscopic examination o corneal cryosections showed widely distributed uorescent nanocarriers, rom the anterior tothe posterior part o the cornea o the GP(+) group, and higher uorescence intensity in the GP(+) group was also observed. Inconclusion, GPs as cationic colloidal carriers were efficiently adsorbed on the negatively charged cornea without irritating the eyeso the rabbits and can be retained in the cornea or a longer time. Tus, GPs(+) have a great potential as vehicles or ocular drugdelivery.

1. Introduction

Te eye poses unique challenges or drug delivery. Te mainobjective o ocular therapeutics is to provide and maintainadequate concentration o the drug at the target site. Mostocular diseases are treated with topical application o solu-tions administered as eye drops. Te major disadvantages othis dosage orm include (i) poor ocular drug bioavailabilitybecause o the anatomical and physiological constraints o

the eye that limit drug retention, (ii) pulse-drug entry withhigh variation in dose, (iii) nasolacrimal duct drainage,which causes systemic exposure, and (iv) poor entrance tothe posterior segments o the eye because o the lens-irisdiaphragm [,]. Te above disadvantages result in clearanceo % o the eye drops within min, and only % o theadministered dose permeates to the eye [].

Most efforts in ocular delivery have been ocused onincreasing the corneal retention o drugs with the nal goal


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Journal o Nanomaterials

o improving the efficacy o treatments or different oculardiseases. Tese attempts include the use o colloidal drugdelivery systems such as liposomes [], nanoparticles [], and nanospheres []. Te results o different studiesshowed the potential o nanoparticles or either gene or drugdelivery or ophthalmic application. Nanoparticles are able to

encapsulate and protect the gene/drug against degradation,improve tolerance, and increase corneal uptake and intraoc-ular hal-lives []. Gelatin nanoparticles (GPs) were selectedor topical delivery because o their unique properties suchas biocompatibility and biodegradability []. Moreover, thesource o gelatin, collagen, which is the major constituent othe corneal stroma, has been used or ophthalmic applica-tions [].

Although several studies have examined the use o GPsor gene/drug delivery [], ew studies have examinedthe use o GPs or ocular delivery. Vandervoort examinedGPs encapsulated pilocarpine or hydrocortisone or topicalophthalmic delivery []. Vandervoot characterized the di-erent orms o GPs and reported the rates o drug releaserom these GPs, but they did not perorm in vitro or invivotests.In vivoadministration o GPs loaded with plasmidDNA showed signicantly higher expression o MUCACin the conjunctiva than that in untreated controls, andnaked plasmid DNA encapsulated in GPs was benecial orophthalmic gene delivery []. Tese results show that GPsmay be effectively used as vehicles or topical administrationto the eyes.

Te cornea and conjunctiva possess negative suracecharges, and it is expected that cationic colloidal nanoparti-cles may penetrate through the negatively charged ocular tis-sues more efficiently than anionic carriers []. o determinethe importance o these characteristics in the interactiono nanoparticles with the cornea, we prepared GPs with apositive and negative charge or ocular delivery. Te GPs withdifferent charge were selected or ocular drug delivery. Weexamined the particle size, polydispersity index (PDI), shape,and surace charge and cytotoxicity o the GPs. Fluorescentlylabeled GPs were used inin vitroandin vivoexperiments toobserve the distribution o the particles in the eyes o rabbits.In addition, the central corneal thicknesses and intraocularpressure (IOP) o rabbits were also examined to conrm theinuence o nanoparticles in rabbit eyes.

2. Materials and Methods

.. Reagent and Chemicals. Gelatin type A (derived romporcine skin, bloom ), gelatin type B (derived rom bovineskin, bloom ), % glutaraldehyde (GA) solution, andacetone were purchased rom Sigma-Aldrich (MO, USA).Dulbeccos modied Eagles medium (DMEM)/F ( : ),etal bovine serum (FBS), insulin, trypsin-EDA, peni-cillin/streptomycin, and phosphate-buffered saline (PBS)wereobtained rom Gibco/BRL (MD, USA);epithelialgrowthactor (EGF) was acquired rom Pepro ech (Rocky Hill, NJ,USA).etramethylrhodamine succinyl (AMRA-NHS)esterand rabbit anti-zona occludens (ZO-) polyclonal antibodywere obtained rom Invitrogen (CA, USA). Te Quick Cell

Prolieration Assay Kit II was got rom BioVision (CA, USA).A Live/Dead Kit was purchased rom Molecular Probes (OR,USA). Single-well cell inserts (PE) were obtained romMillipore (MO, USA). All other chemicals were o reagentgrade and obtained rom Sigma-Aldrich.

.. Preparation of GPs. Te GPs were prepared by a two-step desolvation method as described previously with somemodications [, ]. ype A and type B gelatin solution( wt%) initially underwent desolvation by addition o excessquantity o acetone. Ten, the gelatin deposited was redis-solved in water. Te pH o the type A gelatin solution wasadjusted to . and that o type B was adjusted to . Acetonewas added in a dropwise manner to orm nanoparticles.At the end o the process, L o % GA solution wasused as a crosslinking agent or preparing nanoparticles, andthe solution was stirred or h at rpm. Te remainingorganic solvent was evaporated using a rotary evaporator(EYELA, okyo, Japan), and the resultant nanoparticles were

stored at

C or urther examination.

.. Characterization and Measurement of Different Parame-ters of the GPs. Te size and zeta potential o the GPs wereanalyzed using photon correlation spectroscopy (PCS) andlaser Doppler anemometry, respectively, using a Zetasizer, HS (Malvern Instruments, UK). Each batch was ana-lyzed in triplicate. Te morphology o the nanoparticles wasobtained by scanning the dried particles deposited on a atsurace with a cantilever probe model AC (Olympus,USA) using tapping mode in an atomic orce microscope(AFM; Asylum Research, MFP-DM, USA).

.. Human Corneal Epithelial Cells Culture. Te SV-immortalized human corneal epithelial (HCE) cell line waskindly gifed by Dr. Ko-Hua Chen (aipei Veterans Gen-eral Hospital, aiwan). Te HCE cells were cultured inDMEM/F- supplemented with % FBS, U/mL peni-cillin, . mg/mL streptomycin, ng/mL EGF, .% DMSO,and g/mL insulin. Te cells were cultured at C in a %CO

2-% air atmosphere. Media were changed every other

day, and the cells were observed daily under a phase contrastmicroscope.

... Evaluation of Cytotoxicity of GPs. Te cytotoxicity othe GPs was examined in the HCE cells using the Quick Cell

Prolieration Assay Kit II (BioVision). Te cells were seededonto -well plates ( 3 cells/well) about h beore theexperiment. Te HCE cells were incubated with differentconcentrations o the GPs ( to . g/mL) or h. Ten,the culture medium was discarded, and . mL water-solubletetrazolium- (WS-) working solution was added to eachwell. WS- is reduced by dehydrogenases in the livingcells to produce a yellow colored product (ormazan). Aferincubation or h, L o the working solution was quanti-tatively assessed using a SpectraMAXM spectrophotometer(Molecular Devices, CA, USA) at a wavelength o nm.Te reerence wavelength was set at nm. Te cells werestained with a live/dead stain (Molecular Probe) to observe


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cell viability. Te live cells emit green uorescence, and thedead cells emit red uorescence. Images were acquired usingan inverted uorescence microscope (Nikon, iS, Japan) andwere analyzed using Nikon NIS Element sofware.

... Evaluation of ransepithelial Electrical Resistance.

About 5

HCE cells/cm2

were seeded on PE insertswith a .-m pore size (Millipore, MA, USA), and themedium was replenished every other day. Resistance acrossthe insert membrane was measured using the SX electrodeset (World Precision Instruments [WPI], Florida, USA). Tetransepithelial electrical resistance (EER) o cells grownon lters was measured with an epithelial voltohmmeter(EVOM, WPI). Cells were used only i their EER was more

than V/cm2. Te suspension o GPs ( g/mL) wasadded into the media o the insert well. Te electrode setwas inserted in both the chambers or the indicated times.Te EER was calculated rom the measured resistance andnormalizedusing the area o the monolayer (ohms per square

centimeter). Te background EER o blank insert lterswas subtracted rom the EER o the cell monolayers. Chi-tosan nanoparticles (CNP) were used as the positive controlbecause o their capacity to disrupt the tight intercellular

junctions []. Te size o the CNP was about nm andtheir zeta potential showed a positive charge ( mV).

... Western Blotting. Te HCE cells were lysed to extractthe cellular protein, and their absorbance was measuredat OD / nm beore use. Equal amounts o protein(approximately g) were separated using % sodiumdodecyl sulate polyacrylamide gel electrophoresis. Ten,the proteins were transerred onto nitrocellulose membrane,

and the membranes were blocked in % nonat powderedmilk in ris-buffered saline (BS) and .% ween. Temembranes were incubated with the primary antibody (ZO-at : overnight at C) ollowed by incubation with theappropriate secondary antibodies (horseradish peroxidase[HRP]-conjugated anti-rabbit antibody at : , or hat room temperature). -ubulin was used as the internalcontrol. Bands were visualized using an enhanced chemi-luminescence reagent and exposed to a Fujilm LASIntelligent Dark Box and captured digitally.

... Cellular Uptake Study. GPs with positive or negativecharge were labeled with red uorescence via being conju-

gated with AMRA-NHS ester (Invitrogen) according to themethod described by the manuacturer. Te concentration othe uorescent dye (AMRA) in the GPs ( g/mL) was. g/mL. In addition, we examined the culture mediumwith the dye concentration equal to that in the aqueousormulation. Te uorescent GPs ( g/mL) were cul-tured with HCE cells or h; subsequently, the mediumwas removed, and the cells were washed twice using PBS.Subsequently, . mL o cell lysis solution ( mM ris-HCl,pH ., mM NaCl, and .% riton X-) was added tothe cell pellets, and they were maintained or . h on ice withrequent vortexing. Ten, the cells lysate was collected intoEppendor tubes and centriuged at , rpm or min.

Te cell suspension ( L) was added to a -well plateand the OD was measured at an excitation wavelength o nm and an emission wavelength o nm by the micro-plate spectrophotometer (SpectraMAXM) under uores-cence mode.

.. Preliminary Animal Study. Male New Zealand rabbitsweighing .. kg andwith no signs o ocular inammatoryor gross abnormalities were used. Te in vivo experimentalprotocol was approved by the Institutional Animal Care andUse Committee o the aipei Medical University (IACUCApproval No. LAC--). Te animals were housed instandard cages in a light-controlled room andwere given oodand waterad libitum. We used rabbits or measurement ateach time point, andduring the experiments, the rabbits wereallowed to move their heads reely, and their eye movementswere not restricted.

... In Vivo olerance. Positively charged GPs conjugatedwith the uorescent dye (GP [+] AMRA) were used in thisstudy. We administered L o sterilized GP(+) AMRA inthe lower conjunctival sac o the right eye o rabbits. Te rab-bits simultaneously received L o AMRA in PBS in theirlef eye. Te same volume o PBS was administered to anothergroup o rabbits as control. Tis irritation test was perormedusing a clinical evaluation scale o (absence) to (highest)o discomort, discharge, cornea/conjunctival chemosis, orredness as described in able using a modication othe scoring system established in the Organizationor Economic Cooperation and Development guidelines orocular irritation testing [,]. Te test was perormed on eyes o each group; the test was perormed in eyes in thePBS-treated (control) group. Each animal was observed andtested at ., , , and h afer instillation.

... Clinical Observations. At each study point, we mea-sured the intraocular pressure (IOP) using a Schiotz tonome-ter (AMANN Ophthalmic Instruments, Liptingen, Germany)calibrated according to the manuacturers instructions. Fordetermination o IOP, readings were taken on each eyealternating between the lef and right eyes, and the meanwas calculated []. Central corneal thickness (CC) wasdetermined using an ultrasonic pachymeter (DGH echnol-ogy, Exton, PA, USA) with a hand-held solid probe [].During the measurements, the probe tip o the pachymeterwas held perpendicular to the central cornea. Averages o

readings were recorded. An ophthalmic table slit lamp(opcon Medical Systems Inc., NJ, USA) was used to observeand record the anterior segment. Te rabbits were killed h afer administration o the eye drops. Te eyeballs wereharvested and xed in .% ormaldehyde.

... Fluorescence Quantication. Te rabbits were killed at., , , and h afer the last instillation. Eyeballs wereharvested and cleaned using PBS. Fluorescent GPs in theeyes were quantied using an in vivo imaging system (IVISImaging System Series; Xenogen, USA). Te relativeintensity o uorescence in the eyes was equivalent to theconcentration o uorescent nanoparticles. Te uorescence


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F : Morphology and size o charged GPs with (a) positive or (b) negative charge. Image acquired by atomic orce microscopy.

: Size and zeta potential o gelatin nanoparticles ( = 3).

Size (nm) Zeta (mV)

GPs(+) 180.6 45.7 33.4 10.9GPs() 230.7 84.6 44.2 7.2

intensity o the PBS-treated group was used as the back-ground value. Te quantied area was restricted to thecornea, and the uorescein signal was calculated ( = 5).

... Distribution of GPs in the Cornea Observed Using

Confocal Laser Scanning Microscopy. Afer the uorescenceintensity was quantied, the cornea was excised rom theeyeball and separated into sections. One section wasdirectly mounted on a glass slide and examined under amicroscope without additional processing o the tissue. A -m cryosection was prepared using the other section romthe apical to the lateral end o the cornea. All cornea sampleswere analyzed with a conocal microscope (Nikon, A, Japan).

.. Statistical Analysis. Experiments were perormed at leastin triplicate, andthe results were reported as mean standarddeviation (SD). All data were analyzed with the Studentst-test or one-way analysis o variance (ANOVA). Statistical

signicance was considered at a level o < 0.05.

3. Results and Discussion

In this study, we prepared charged GPs and perormed invitroand in vivostudies. Te rabbit cornea model was usedto determine the retention o the cationic GPs because othe similarity o this model with that o the human cornea[,].

.. Characterization of GPs. GPs can be prepared using typeA or type B gelatin to obtain positively or negatively chargednanocarriers (able ). Te size o GPs prepared using type

A gelatin was approximately 180.6 45.7 nm. Te size othe negatively charged GPs (prepared using type B gelatin)was 230.7 84.6 nm; these nanoparticles were larger andmore widely distributed than GPs prepared using type Agelatin. Te zeta potential o GPs prepared using type Aand type B gelatin was 33.4 10.9 mV and 44.2 7.2 mV(able ). Te nanoparticles prepared using type A gelatinhad a positive surace charge and were abbreviated as GP(+),and those prepared using type B gelatin were negativelycharged and were abbreviated as GP(). Te nanoparticleso both types observed under the AFM showed a smoothand ball like structure (Figure ). Te particle size was about nm, which was consistent with the ndings o photoncorrelation spectroscopy (PCS). ype A and type B gelatinwere prepared using by different processes by extractinggelatins rom collagen []. Te amount o ree carboxyl oramino groups was different in both types o gelatin. At pH, however, type A gelatin has a positive net charge, whiletype B gelatin is negatively charged [, ]; thus, the zeta

potentialo these gelatins may also be different. Te positivelycharged GPs (GP+) may have electrostatic attraction withthe negatively charged corneal epithelial cells, which is morepreerred in ocular drug delivery.

.. Cytotoxicity of GPs. An important aspect o the devel-opment o new carrier or drug/gene delivery is its saety ointeraction with the target cells. Te biocompatibility o thenewly developed materials should be examined to determinetheir potential or ophthalmic use. In this study, we evaluatedthe cytotoxicity o GPs in the HCE cell line by measuringtheir metabolic activity. Te percentage o viable cells in thetreated group versus nontreated group (culture medium) is


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: Grading system o the macroscopic signs in thein vivotolerance study or the colloidal system tested [].

Grade Discomort Cornea Conjunctiva Discharge Lids

No reaction No alterations No alterations No discharge No swelling

Blinking Mild opacity Mild hyperemia

Mild edemaMild discharge without

moistened hair Mild swelling

Enhanced blinking

Intense tearingVocalizations

Intense opacity

Intense hyperemia

Intense edemaHemorrhage

Intense discharge withmoistened hair

Obvious swelling

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F : Results o WS- assays o human corneal epithelium (HCE) cells afer incubation with kinds o gelatin nanoparticles or h.(GP+, GPs with positive surace charge; GP, GPs with negative charge. Data were analyzed using the Students t-test and are presented asmeanstandard deviation (SD); = 6, < 0.05).

shown in Figure . No signicant difference was observedin cell viability even afer treatment with g/mL o GPs

or h. Te images o HCE cells labeled using the live/deadstain are shown inFigure ; the live cells emit green uoresceand the dead cells emit red uoresce. Large percentage olive cells was observed in the control group (Figure (a)),and nearly all the HCE cells were viable afer coculturingwith GP(+) or GP() or h (Figures(b)and (c)). HCEswere viable and only a ew dead cells were observed aferculturing with GPs, which indicated that GPs had adequatesaety or application to the ocular surace. de la Fuente et al.cultured HCE cells with hyaluronic acid-CNP or h andshowed that this treatment had no effect on cell viability[]. Te viability o HCE cells treated with cationized gelatinnanovector hybrid with chondroitin sulate or dextran sulate

or h was not signicantly different rom that nontreatedHCE cells []. Tese results indicate that GPs are not toxictoHCE cells afer short-term or long-term exposure.

.. Alteration in the EER across the ight Junction in HCECells by GPs reatment. Te presence o tight junctionsbetween epithelial cells prevents the ow o the uids andthe movement o molecules and ions between cells [].Te epithelial membrane provides a signicant barrier to theree diffusion o substances rom the cornea to the anteriorchamber. Te barrier integrity o these monolayers can bemeasured directly by measuring the EER o HCE cells.Te EER o HCE monolayers cultured with charged GPs

(GP+/GP) slightly increased to % .% (% againstinitial) and then returned to normal (Figure (a)). No signi-

cant difference was observed in the EER o HCEcells treatedwith GPs(+) or GP() afer h. However, cells treatedwith CNP showed a marked decrease in the EER (%decrease), which showed that the barrier integrity o the HCEmonolayer was changed by CNP treatment. Te CNP-treatedcells showed losso ZO-, but no variation in the GPs groups(Figure (b)). Chitosan has been widely used or ocular drugdelivery [, ] because o its mucoadhesive property.Chitosan disrupts the tight intercellular junctions and resultsin loss o membrane-associated ZO-, thus increasing thepermeability o the epithelium []. Te anterior part o theeye is constantly exposed to the external environment andthus is vulnerable to a wide range o microorganisms; urther,

its moist mucosal surace makes the cornea particularlysusceptible to attack. Te barrier to avoid microorganisminvasion depends on the integrity o the tight junctions inthe cornea. Te tight junctions between the neighboringepithelial cells prevent the ree diffusion o hydrophilicmolecules across the epithelium by the paracellular route[]. However, CNP increases the drug concentration in thecornea via intracellular (uptake by the cells) and intercellular(opening the tight intercellular junction) routes []. Previousstudyshowed that the tight junction reclosed maybe impededby the unremoved chitosan residue on the surace o theCaco- cells []. Tereore, a similar phenomenon may beobserved in the corneal epithelial cells causing continuously


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(a)

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F : Fluorescentphotomicrographs o humancorneal epithelial (HCE) cells cultured with (a) culture medium, (b) GP(+), and(c) GP()at a concentrationo g/mL or h. Te polyanionic dye calcein-AM is well retained within the live cells, which produced intense uniormgreen uorescence in the live cells. (Magnication: x); GP: gelatin nanoparticles.

open o the tight junction. Tereore, a risk or using CNPor ocular drug delivery is increasing the microorganism

invasion to the cornea via disruption o cornea tight junction.But, there is no risk or tight junction disruption by GPs.

.. Intracellular Content. We examined the intracellular acc-umulation o the charged GPs in the HCE cells. We examinedinternalization o uorescence-labeled GPs by measuring theuorescence in the cell lysates. Cationic or ionic GPs con-

jugated with AMRA were added into the culture medium.Te intracellular uorescence o the cell lysates in the GP(+)group at, , and min was higher than that o the GP()(Figure ). Afer min, the OD value o the GP(+) groupwas much higher than that o the GP() group ( < 0.05).Tis nding is consistent with previous study, which showed

that cationic nanoparticles could increase the stability o thenanoparticle system and improve the interaction between

the particles and the eye surace and thus increase thetransection efficiency [,].

.. olerance and Clinical Evaluation. Gelatin is commonlyused in the preparation o capsules, and GPs are widelyinvestigated or drug/gene delivery. However, ew studieshave examined the saety and tolerance o GPs or oculardrug delivery. We perormed an irritation test on rabbitsafer single instillation o L o GPs ormulation. Te eyetreated using PBS was used as a control. Each animal wasobserved at ., , , and h afer instillation. An indexo overall irritation (able ) was calculated by summing upthe total clinical evaluation scores over the observation time


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F : Te transepithelial electrical resistance (EER) assay showed recovery o human corneal epithelial (HCE) cell layer barrier afercoculturing with gelatin nanoparticles (GPs), but not in chitosan nanoparticles (CNP). = 5standard error o mean (SEM), < 0.05. (b)Western blot analysis o zonula occluden- (ZO-) expression in HCE cells afer treatment with different nanoparticles.

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F : Nanoparticles uptaken by the human corneal epithelial (HCE) cells were evaluated by measuring the uorescence intensity o thecell lysate. = 6standard error o mean (SEM),

< 0.05.

: Grading system o macroscopic signs in thein vivotolerance study o the gelatin nanoparticles.

Control Free AMRA GP(+) AMRA

ime . h . h . h . h . h . h . h . h . h . h

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= 3.GP: gelatin nanoparticles; AMRA: tetramethyl rhodamine.


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F : Te appearance o rabbit eyes topically treated with L o ree tetramethyl rhodamine succinyl (AMRA) solution: (a) . hand (b) h afer treatment; treated with GP(+)AMRA solution ( L): (c) . h and (d) h afer the application.

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F : (a) Measurements o central corneal thickness (CC) and (b) intraocular pressure (IOP) afer treatment with eye drops containingAMRA solution or GP(+)AMRA. An asterisk indicates statistically signicant differences (

< 0.05; = 5) compared to control (PBS-

treated rabbits, = 3).


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F :Ex vivouorescence imaging o the eyes o rabbits treated with uorescent dye or different time periods: (a) AMRA solution and

(b) GP(+)AMRA. = 5standard error o mean (SEM),

< 0.05.

EP

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F : ((a), (b)) are images rom cryosections o the cornea treated with the ree dye and GP(+) uorescence dye. Free dye: AMRA/PBSsolution; GP(+) dye: GP(+) with AMRA conjugation (red). Scale bar: mm. EP: corneal epithelium; S: corneal stroma; EN: cornealendothelium. Afer treatment or . h.

points; the results are shown in able . Very slight rednesso the conjunctiva was observed in the eyes treated with reeAMRA solution and in GP(+) AMRA-treated eyes at h,but no chemosis was observed afer treatment in other groupsand at other time points. No differences were observed in theocular tissue o rabbits treated with ree and GP(+) AMRAafer . and h (Figure ). reatment with GP(+) was sae

and caused no irritation to the eyes o the rabbits. Previousstudies have shown that the rabbit eye is more sensitive thanthe human eye and has a longer time or epithelial repair[,]. Tereore, it is reasonable to expect that charged GPsare well tolerated by the human eye.

One o the risk actors involved in eye disease is increasedIOP, which leads to apoptosis and loss o retinal ganglioncells []. Tereore, we examined the changes in the IOPand corneal thickness afer eye drop treatment to conrmthe saety o GPs or ocular drug delivery. Te effects oinstillation o ree AMRA and GP(+) AMRA eye dropson corneal thickness and IOP in rabbits are shown inFigure . Compared to the control groups (328 21 m), the

groups treated with ree AMRA and GP(+) AMRA eyedrops showed a decrease in the corneal thickness afer . h(297 4 m;292 6 m) and h (304 5 m;290 5 m)(Figure (a)). Afer h o treatment, the corneal thicknessin the treated group was almost the same as that in thecontrol group. Te mean baseline o IOP values ranged romtommHg(Figure (b)). Te IOPdecreased immediately

afer treatment with PBS. Te IOP returned to the baselinelevel within h. In addition, the IOP decreased at h evenafer treatment with ree AMRA and GP(+) AMRA. How-ever, the IOP in these groups returned to the normal rangeafer h (Figure (b)). Te corneal thickness and IOP didnot change signicantly afer treatment with GP(+) AMRAsuspension.

.. Fluorescence Examination to Determine the Distributionof GPs in the Eyes. Te amount o uorescent nanoparticlesin the cornea at different time points acquired using theIVIS spectrum imaging system is shown in Figure . Tenumber o uorescent spots obtained afer treatment with


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GP(+) AMRA wasgreater than that obtained treatment withree AMRA/PBS at ., , , and h. Te accumulationo the uorescent dye differed signicantly between the reeAMRA and GP(+) AMRA treated group at h afer treat-ment. Afer IVIS examination, the cornea was removed anda cryosection was prepared or examination under a conocal

microscope. Te distribution prole o the uorescent dye inthe cornea o rabbits afer administration o the eye dropsis shown inFigure . Te cross-section o the cornea romthe epithelium, stroma to endothelium layer, was observedunder the same magnication. Te cornea treated usingthe ree AMRA solution showed a weak uorescent signallocated in the posterior region (Figure (a)). Te corneatreated with GP(+) AMRA showed a strong uorescentsignal in the entire cornea and (Figure (b)). Moreover, theuorescence quantication o the cornea treated with GP(+)AMRA increased by -old compared to that treated withAMRA/PBS solution, which indicated that the dye encapsu-lated in the GPs could be retained in the cornea or a longertime and was distributed uniormly across the entire cornea.Solid lipid nanoparticles (SLNs) and CNP are retained ora longer time on the corneal surace probably because otheir small size, and urther characterization o nanoparticleswould help in determining the transcorneal absorption [,]. Hyaluronic acid coated poly--caprolactone nanospheresachieved high levels o cyclosporine A (CyA) in the cornea,which was -old higher than that was achieved with CyAsolution in castor oil []. In our study, the levels o GPs inthe corneas treated with GP(+) AMRA were higher thanthose in the cornea treated with ree AMRA at each timepoint, which indicated a longer retention time ( h) o GP(+)AMRA compared to that o ree AMRA. Our in vivoresultsmight be explained on the basis o the prolonged retentionin the precorneal area and cornea because o the small sizeo GP(+) ( nm) and also in the uptake/internalization oGP(+) into the corneal epithelium.

4. Conclusion

Te aim o this study was to conrm whether cationic GPscould be used or topical application, and this was examinedin rabbit eyes. Positively charged GPs were prepared with asize o about nm. GPs are nontoxic to HCE cells and hadno inuence on the tight intercellular junctions. Te cornealthickness slightly decreased . h afer treatment and then

returned to normal. Te IOP showed variation in the normalrange afer treatment with GPs. GPs showed retention othe uorescent dye in the cornea or the prolonged period,which is benecial to maintain the dose in the therapeuticrange. Tereore, dye/drug/gene encapsulated in cationic GPsnanoparticles is promising new medicines or ocular disease.

Acknowledgments

Tis work was supported in part by a grant rom theaipei Medical University Startup Grant (MU-AE-B)and National Science Council, aiwan (NSC --E--).

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