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Cyclosporine A loaded mucoadhesive nanoparticle eye drop formulation enhances treatment of experimental dry eye in mice using a weekly dosing regimen Shengyan Liu, Matthew D. Dozois, Chu Ning Chang, Aaminah Ahmad, Deborah Ng, Denise Hileeto, Huiyuan Liang, Matthew-Mina Reyad, Shelley Boyd, Lyndon Jones, and Frank X. Gu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00445 • Publication Date (Web): 02 Aug 2016 Downloaded from http://pubs.acs.org on August 7, 2016

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Molecular Pharmaceutics

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Prolonged ocular retention of mucoadhesive

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nanoparticle eye drop formulation enables treatment

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of eye diseases using significantly reduced dosage

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Shengyan Liu1,2, Matthew D. Dozois1,2, Chu Ning Chang1,2, Aaminah Ahmad1,2, Deborah L. T.

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Ng1,2, Denise Hileeto3, Huiyuan Liang4, Matthew-Mina Reyad4, Shelley Boyd4, Lyndon W.

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Jones2,3, and Frank X. Gu1,2 *

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1

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2

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3

Waterloo Institute of Nanotechnology, University of Waterloo, Waterloo, Canada

Centre for Contact Lens Research, School of Optometry and Vision Science, University of

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Department of Chemical Engineering, University of Waterloo, Waterloo, Canada

Waterloo, Waterloo, Canada 4

Keenan Research Centre for Biomedical Science, St. Michael’s Hospital, Toronto, Canada

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Keywords: Mucoadhesive nanoparticles, ocular retention, drug delivery, phenylboronic acid, dry

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eye

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ACS Paragon Plus Environment


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ABSTRACT

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Eye diseases, such as dry eye syndrome, are commonly treated with eye drop formulations.

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However, eye drop formulations require frequent dosing with high drug concentrations due to

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poor ocular surface retention, which leads to poor patient compliance and high risks of side

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effects. We developed a mucoadhesive nanoparticle eye drop delivery platform to prolong the

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ocular retention of topical drugs, thus enabling treatment of eye diseases using reduced dosage.

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Using fluorescent imaging on rabbit eyes, we showed ocular retention of the fluorescent dye

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delivered through these nanoparticles beyond 24 hrs while free dyes were mostly cleared from

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the ocular surface within 3 hrs after administration. Utilizing the prolonged retention of the

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nanoparticles, we demonstrated effective treatment of experimentally-induced dry eye in mice by

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delivering Cyclosporine A (CsA) bound to this delivery system. The once a week dosing of

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0.005 to 0.01% CsA in NP eye drop formulation demonstrated both the elimination of the

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inflammation signs and the recovery of ocular surface goblet cells after a month. Thrice daily

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administration of Restasis® on mice only showed elimination without recovering the ocular

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surface goblet cells. The mucoadhesive nanoparticle eye drop platform demonstrated prolonged

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ocular surface retention and effective treatment of dry eye conditions with up to 50 to 100 fold

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reduction in overall dosage of CsA compared to Restasis®, which may significantly reduce side

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effects and by extending the inter-dosing interval, improve patient compliance.

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INTRODUCTION

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Dry eye syndrome (DES) is defined as a multifactorial disease of the tears and ocular surface

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that results in symptoms of discomfort, visual disturbance, and tear film instability with potential

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damage to the ocular surface.1 DES is also accompanied by increased osmolality of the tear film


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and inflammation of the ocular surface. Cyclosporine A (CsA, an immunosuppressant agent)

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0.05% ophthalmic emulsion (Restasis®, Allergen Inc., Irvine, CA) is the first commercially

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available eye drop formulation that showed an increase in natural tear production in dry eye

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patients, in part by acting to reduce both inflammation and concurrent loss of the conjunctival

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epithelium and goblet cells.2 Eye drops, such as Restasis®, are the most popular method of

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delivering therapeutics to the eye due to their convenience and non-invasiveness, but their

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biggest challenge is rapid clearance due to tear dilution and turnover: more than 95% of the

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administered drugs are cleared before they reach their intended target.3 Hence, eye drop

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formulations are often administered frequently (twice daily for Restasis®, but up to four times

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for many topical drugs) at high doses so that the drug concentrations may reach the therapeutic

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window. However, the high overall dosage of the administered drugs results in increased side

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effects: approximately 25% of the people in the phase III safety evaluation of Restasis® reported

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experiencing one or more adverse effects such as burning, stinging and foreign body sensation

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on the ocular surface.2, 4 Since DES requires constant long-term treatment of its symptoms and

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signs, reducing the dosage of CsA using a more efficient drug delivery platform may likely

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reduce side effects and improve patient compliance, without compromising its therapeutic

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efficacy. Several studies in the past have utilized different delivery platform to improve the

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solubility of the CsA in aqueous formulations as well as enhancing their corneal penetration, but

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reduced dosage was not demonstrated due to the lack of prolonged ocular retention of the CsA.5

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In addition, lower administration rates would reduce the likelihood for adverse reactions to the

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preservatives that are commonly included in topical drugs and which are a common cause of

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ocular surface damage.6-9



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Nanoparticle (NP) as drug carriers have been proposed to address the challenges associated

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with conventional eye drop delivery methods.10-13 By encapsulating drugs as their cargo, NPs

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may increase the concentration of drugs in the formulation, control the release rates of the drugs,

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and improve corneal retention by targeting the ocular surface.14, 15 The controlled release of the

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drugs from the NP drug carriers may also reduce the total amount of drugs exposed on the ocular

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tissue at any given time, thus reducing the risk of side effects. Moreover, the small size of the NP

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(particle sizes ≤ 10 µm) eliminates discomfort experienced by users from administration of larger

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particles.16 The NP formulation may also be tuned to achieve transparency and viscosity similar

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to that of water. A number of polyester based polymers, such as poly(lactic acid) (PLA),

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poly(glycolic acid) (PGA), poly(ε-caprolactone) (PCL), and their copolymers have been

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explored as suitable materials for preparing a NP drug delivery platform for targeting eye

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diseases.17-23 These NPs have been coated with polyethylene glycol (PEG) to improve their

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colloidal stability.24-30 Recently, we developed an amphiphilic block copolymer composed of

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poly(D,L-lactic acid) and dextran (PLA-b-Dex) to formulate NP drug carriers.31 Dextran

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provides a unique advantage over PEG-based materials due to its greater hydrophilicity and

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significantly higher density of functional groups for surface modification. Furthermore, dextran

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based NPs have previously demonstrated superior colloidal stability compared to PEG based

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NPs.32 Dextran has three hydroxyl groups per monomer, whereas PEG only has one functional

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end group per chain: increased functional groups improves the efficiency of surface modification

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and consequently provides greater control over the surface properties of the NPs. Previously, we

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modified the surface of PLA-b-Dex NPs with phenylboronic acid (PBA) molecules to achieve

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mucoadhesion through covalent linkage between PBA and the cis-diol groups of carbohydrates

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abundant on the ocular mucous membrane.33 By covalent attachment, PBA-modified NPs


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provide a greater affinity towards mucous membranes compared to the more commonly studied

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mucous-targeting molecules, such as chitosan, which rely on physical interaction with the

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mucous membrane.34-38 Since the NPs target the surface mucous membrane, the rate of clearance

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of the NPs is likely reflected by the turnover rate of the ocular mucous membrane. We

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previously demonstrated significantly improved mucoadhesive properties using PBA-modified

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NPs, compared to Chitosan-based or thiol-based NPs, and a sustained release of CsA for up to 5

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days. We further demonstrated that the CsA-loaded NPs were biocompatible on rabbit eyes, and

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effective in treating dry eye conditions in a short term study on mice.39 We hypothesize that the

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mucoadhesive and controlled release properties of the nanoparticle drug carriers provide a drug

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delivery platform for long term treatment of dry eye using significantly reduced dosage of the

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drug, while maintaining treatment efficacy. The objective of this study is to investigate whether

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the CsA-loaded PBA-modified NPs are able to treat long-term experimental DES using once-a-

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week dosing.

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In this study, experimental DES was induced in mice and nanoparticle formulations with

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difference doses of CsA were applied once a week to treat the dry eye signs. Dry eye treatment

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efficacy was evaluated by measuring for tear production and clearance, and also by evaluating

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the inflammation and ocular surface recovery using histopathology analysis post-euthanasia.

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The treatment efficacy of the CsA loaded PBA modified NP formulations were compared with

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Restasis® applied as a topical drop.

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EXPERMENTAL SECTION

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Materials. Acid-terminated poly(D,L-lactide) (PLA; Mw ~20) were purchased from

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Lakeshore Biomaterials (USA) and washed with methanol to remove monomer impurities.

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Dextran (Dex; Mw ~10 kDa), 3-Aminophenylboronic acid monohydrate (PBA), sodium



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periodate (NaIO4), glycerol, sodium cyanoborohydride (NaCNBH3), sodium fluorescein, N-

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acetylneuraminic acid (sialic acid), indocyanine green (ICG) and Cyclosporine A were purchased

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from Sigma Aldrich (Canada). Cyanine 7.5 hydrazide was purchased from Lumiprobe (USA).

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Nanoparticle formulation, and encapsulation and in vitro release of Cyclosporine A. The

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amphiphilic block copolymer poly(D,L-lactide)-b-dextran (PLA-b-Dex) polymer was prepared

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using a previously reported method.31 NPs were formed by the self-assembly process of the

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amphiphilic block copolymer through a nanoprecipitation method. To achieve mucoadhesive

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properties for long retention of ocular drugs on the corneal surface, we modified the surface of

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the NPs with phenylboronic acid (PBA) in two steps: the hydroxyl groups of the dextran surface

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of the NPs were oxidized in the presence of NaIO4, and the aldehyde groups were then reacted

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with the amino groups of PBA using reductive amination in the presence of NaCNBH3.33

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The encapsulation of raw CsA in the PBA-modified NPs (PLA-b-Dex-g-PBA NPs) was

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performed using a nanoprecipitation method. 1 ml of DMSO containing polymer (~ 7 mg/ml)

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and CsA (varied concentration) was slowly added into 10 ml of Millipore water under mild

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stirring for self-assembly of NPs carrying the drugs. The NP-drug mixture was then syringe

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filtered (pore size = 200 nm) to remove NP or drug aggregates, and dialyzed against water to

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remove some of the free drugs and DMSO from the mixture. The sizes of the nanoparticles were

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determined using dynamic light scattering (DLS) technique. The amount of CsA in the final

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mixture was determined using High-performance liquid chromatography (HPLC; C18 HPLC

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column, ACN/H2O 75:25 as the mobile phase with UV-absorption detection at 210 nm).

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The CsA in vitro release from the NPs was measured using the method reported previously.33 8

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mL of syringe filtered NP-CsA suspension was injected into a Slide-a-Lyzer Dialysis cassette

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(MWCO = 20 kDa, Fisher Scientific) and dialyzed against 300 mL of simulated tear fluid (0.654


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g of NaHCO3, 2.04 g of NaCl, 0.0189 g of CaCl2, 0.414 g of KCl in 300 mL of Millipore water)

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at 37 °C. 1 mL of the release medium was extracted at each predetermined time points to

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quantify the amount of CsA using HPLC, while 1 mL of fresh simulated tear fluid was added to

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the release medium.

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In vitro interaction between PBA modified NPs and sialic acid. To evaluate the

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mucoadhesive properties of PBA-modified NPs, we studied the covalent interaction between the

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PBA molecules on the surface of the NPs with sialic acid (SA) molecules which are abundant on

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the mucous membrane. The covalent complexation between PBA and SA quenches the intrinsic

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fluorescence of the PBA molecules. Thus, we can quantify the extent of covalent interaction

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between PBA and SA by analyzing the relative fluorescence intensities of PBA in the presence

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of SA. PBA-modified NPs suspensions were mixed with sialic acid (SA) solutions to achieve

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constant final concentration of PBA-modified NPs (50 µg/ml) with varying SA concentrations

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(0, 0.01, 0.02, 0.04, 0.08, 0.12, 0.16, and 0.32 mM). The mixtures were vortexed for 30 seconds

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before measurement with a spectrofluorometer (type LS-100, Photon Technology International,

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Canada). The samples were excited at 298 nm, and the emission scan from 310 to 450 nm was

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obtained for each sample. The relative fluorescence data was then analyzed to determine the

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Stern-Volmer binding constant, KSV, using the Stern-Volmer equation 40: ‫ܫ‬଴ൗ ‫ = ܫ‬1 + ‫ܭ‬ௌ௏ × ሾܳሿ

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where I0 is the fluorescence intensity of the PBA-modified NPs sample without SA ([SA] = 0

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mM), I is the fluorescence intensity of NPs with SA, and [Q] is the concentration of the

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quenching agent, SA. The KSV value was determined by calculating the slope of the data.

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Animal studies. All animal studies adhered to the Association for Research in Vision and

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Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research.



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The animal studies were also in compliance with the guidelines of the Canadian Council on

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Animal Care as well as regulations under the Animals for Research Act of Ontario Canada.

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Ethics approval was obtained from the University of Waterloo prior to the start of the study.

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Male New Zealand White (NZW) rabbits (Charles River Laboratories, Canada) with average

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weights of 2.8 to 3.3 kg were used for the ocular surface retention study. Female C57BL/6 mice

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(Charles River Laboratories, Canada) aged 3-5 months were used experimental dry eye treatment

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study. All animals were acclimatized in the animal facility for at least one week prior to

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experimentation. All formulations administered to the mice were sterilized using a sterile syringe

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filters (pore size = 0.2 µm) prior to use.

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Ocular surface retention study on NZW rabbits. We used two different approaches to

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evaluate the ocular retention of the nanoparticle eye drop formulation: first, we evaluated the

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retention of the nanoparticle material by tracking the fluorescent labelled nanoparticle shell

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material on the rabbit eyes, and second, we evaluated the ocular retention of the drugs

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encapsulated in the nanoparticles by using a fluorescent dye as a model drug. In the first study,

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we evaluated the effect of PBA on the ocular retention of the PBA modified NPs. Cyanine with

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hydrazide functional group, a near-infrared fluorescent dye, was conjugated to the oxidized Dex

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through reductive amination, in the similar manner to the PBA conjugation onto Dex. The

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unreacted PBA and cyanine were removed through multiple methanol wash. To evaluate the

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ocular retention of the nanoparticle shell material (i.e. dextran-PBA), we compared cyanine

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labelled dextran (Dex-cyanine – PBA; 25 mg/ml) with cyanine labelled dextran-PBA (Dex-

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cyanine + PBA; 25 mg/ml) using a confocal scanning laser ophthalmoscopy (cSLO; Spectralis®:

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Heidelberg Retinal Angiography 2, Heidelberg Engineering, Germany).


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In the second study, we used indocyanine green (ICG), a near-infrared fluorescent dye, as a

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model drug to evaluate the ocular retention time of a drug delivered through PBA-modified NPs,

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also using cSLO. ICG was encapsulated in the NPs using the same nanoprecipitation method that

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was used to encapsulate CsA. ICG encapsulated NPs were then dialyzed to remove free ICG and

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DMSO. We also evaluated the retention of ICG encapsulated in unmodified NPs to further

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evaluate the effect of PBA on the ocular retention. We compared the ocular retention of free ICG

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or ICG encapsulated in unmodified NPs (NP-ICG – PBA) with ICG encapsulated in PBA

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modified NPs (NP-ICG + PBA).

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NZW male rabbits were sedated with acepromazine (2mg/kg) and briefly anesthetized using a

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mask with 2% isoflurane for 10 minutes for eye drop instillation. For each rabbit, one eye was

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administered with 50 µL of sample formulations (Dex-cyanine + PBA or NP-ICG + PBA) while

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the contralateral eye was administered with 50 µL of control formulations (Dex-cyanine – PBA,

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Free ICG or NP-ICG – PBA). We used cSLO for imaging at baseline before administration and

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at 0, 3, 6, 9 and 24 hrs after the initial administration. After imaging at each time point, rabbits

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were transferred back to their cages to blink normally. To image cyanine or ICG, we used an

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excitation wavelength of 795 nm and an emission wavelength at 810 nm for all images. To

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achieve standardized images, the power and sensitivity were routinely set at 100% and 90 units,

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respectively, and the relative brightness of the ocular surface images were analyzed using

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ImageJ. An eclipse was drawn on the ocular surface to enclose as much area as possible while

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avoiding the bright edges between the corneal surface and the eyelids where dye/particles

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accumulate due to pooling. The average brightness of the area inside the eclipse was measured

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and the background signal was subtracted from it.

All of the means of the fluorescence



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measurements were normalized with respect to the initial mean fluorescence measurements at

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time 0 hr, right after instillation of the eye drops.

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Dry eye induction and nanoparticle administration on C57BL/6 mice. To evaluate the

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efficacy of NPs carrying CsA in treating DES, an experimental murine dry eye model was

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used.41 Transdermal scopolamine patches (Transderm-V, Novartis; 1.5 mg of scopolamine) were

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cut into two pieces and wrapped around the midtails of the mice and secured with surgical tape.

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The patches were replaced every other day through the duration of the study. To simulate a

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desiccating environment, mice cages (open-top) were placed in a fumehood for 1 hr, 3 times a

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day throughout the study. After 4 days of dry eye induction, the mice were divided into 6 groups

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of 5 mice and the administration of various formulations were initiated (Table 1). Note that dry

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eye was not induced in the Healthy group and no drug was administered to that group of mice.

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Saline, Blank NP, NP-CsA 0.005-0.01% and NP-CsA 0.025% were administered once a week (7

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µl; on days 5, 12, 19 and 26). RESTASIS® was given 3 times a day (3 µ) throughout the study

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period.42 Tear production and corneal fluorescein clearance were assessed to evaluate the tear

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restoration as a result of the dry eye treatment. Histopathology analysis was performed to

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evaluate the extent of reduction in tissue inflammation as a direct result of the treatment using

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CsA which is an immunosuppressant agent. On days 5, 12, 19 and 26, before the weekly

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administrations, tear production measurement and corneal fluorescein clearance assessments

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were performed. On the final day (day 33), the mice were euthanized and the ocular tissues

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harvested for histopathology.

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Tear production measurement. Tear production was measured by holding a phenol red

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thread (Zone-quick; White Ophthalmic Supply, Canada) with jeweler’s forceps in the lateral


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canthus of the ocular surface of the mice for 30 seconds. The length of the dyed segment (red) of

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the thread was measured and recorded.

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Corneal fluorescein clearance. Corneal fluorescein staining on mice eyes was observed,

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recorded and photographed with a slit-lamp bio-microscope using a cobalt blue light, 10 minutes

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after the administration of 1 µl of sodium fluorescein solution (10 mg/ml).

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Histopathology analysis. The eyes of the mice were enucleated and collected immediately

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after euthanasia for histopathological evaluation. The entire upper eyelids were also dissected

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and collected for evaluation of the tarsal conjunctiva and the underlying soft tissues. Consecutive

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sections of the entire ocular globe and eyelids were processed for microscopic analysis: after

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initial fixation in 10% neutral buffered formalin, the tissue was embedded in paraffin, serially

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sectioned into 5 µm thick sections, and stained with hematoxylin and eosin (H&E). The

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histological slides were evaluated using bright field microscopy (Leica DM1000, ICC50 HD,

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Leica Microsystems Inc, Canada).

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RESULTS

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Nanoparticle formulation. The nanoparticles were fabricated with approximately 15.2 ± 1.0

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mol% of PBA on dextran monomer. The diameter of the raw nanoparticles (Blank NPs) was 25.4

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± 0.6 nm (Table 2). With encapsulation of CsA, the diameter increased slightly to 26.9 ± 1.1 nm

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(NP-CsA 0.005-0.01%), and to 29.1 ± 0.3 nm (NP-CsA 0.025%).33, 39 From HPLC analysis, the

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NP-CsA 0.025% group contained approximately 1.5 µg of CsA in a 7 µl eye drop. The

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concentrations of the nanoparticles in Blank NPs, NP-CsA 0.005-0.01%, and NP-CsA 0.025%

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were all kept at 0.57 mg/ml (about 4 µg in a 7 µL eye drop) (Table 1). This means that the NP-

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CsA 0.025% group had about the same amount of CsA in a 7 µl drop as that in a 3 µl drop of

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Restasis®. Due to the dosing frequency differences between Restasis® and NP-CsA 0.025%, the



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overall dosage of CsA in NP-CsA 0.025% is approximately 5% that of Restasis® (or 1/21 that of

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Restasis®). Similarly, the NP-CsA 0.005-0.01% group had 0.3 µg of CsA in the 7 µl drop for the

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first two weeks of administration, and 0.6 µg for the subsequent two weeks. Thus, the dosage of

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NP-CsA 0.005-0.01% is approximately 1% that of Restasis® (or 1/105th of Restasis®) for the

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first two weeks, and 2% (2/105th of Restasis®) for the subsequent two weeks. The NP-CsA

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0.025% and NP-CsA 0.005-0.01% showed total in vitro release for up to 4 and 5 days

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respectively (Supplementary Figure S1), similar to our previous study.33

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In vitro interaction of PBA modified NPs with sialic acid. To evaluate the mucoadhesive

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properties of PBA modified NPs, the covalent interaction between PBA and SA, found

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abundantly in mucin, was studied using a spectrofluorometer. As the SA concentration increased

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relative to the concentration of the NPs, the maximum fluorescence intensity of the PBA

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molecules at λem = 376 nm gradually decreased. The relative fluorescence intensities were

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plotted against the varying concentrations of SA in the solution to determine the Stern-Volmer

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binding constant KSV. The calculated value of KSV was (1.38 ± 0.03) × 104 for the interaction

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between the PBA modified NPs and SA.

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Ocular surface retention study on NZW rabbits. To analyze the effect of PBA on the in

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vivo ocular retention of the NPs, Cyanine labelled dextran-PBA, the shell material of the NPs

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and Cyanine labelled dextran were compared using cSLO imaging. PBA modified dextran

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showed clear retention for up to 6 hrs after the initial eye drop administration, whereas the

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unmodified dextran showed negligible amount of retention 3 hrs and 6 hrs after the initial

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administration (Figure 2). Furthermore, ICG was used as a model drug for imaging the corneal

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surface of rabbit eyes using cSLO. Standardized cSLO images (Figure 3) showed that the ICG

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delivered using NPs (NP-ICGs + PBA) demonstrated higher retention of ICG compared to the


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ICG control, which was especially noticeable at the 6 hr time-point (Figure 3): up to 24.9% of

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the initial ICG from NP-ICGs administration was still retained after 6 hrs whereas only about

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4.26 % of the ICG was retained after administration of Free ICG (Figure 4). 24 hrs after the

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initial administration, NP-ICG + PBA retained up to 13.7 % of the initial brightness, whereas

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Free ICG remained near baseline level of 4.01%. The unmodified NPs encapsulating ICG (NP-

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ICG – PBA) showed similar ocular clearance rate of ICG as that of the Free ICG.

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Tear production and corneal fluorescein clearance on C57BL/6 mice. To evaluate the

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efficacy of the CsA loaded NPs in treating DES, tear production measurements and corneal

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fluorescein staining analysis were performed each week right before the dosing of the various

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formulations on mice. All the groups (with the exception of the Healthy control group)

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underwent dry eye induction, which was reflected in the sudden drop of tear production after 4

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days (ie, on day 5) (Figure 5). The Healthy group maintained a relatively stable tear production

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throughout the study except on week 3 (day 26). Interestingly, this slight drop in tear production

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is also reflected on the reduced corneal fluorescein clearance (Figure 6, top row) observed on day

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26, while on all the other measurement time points the fluorescein was rapidly cleared from the

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corneal surface within 10 minutes. The tear production for all the treatment groups remained at

281

or below half of the initial tear production rate throughout the duration of the study. Only the

282

NP-CsA 0.025% group was able to significantly increase its tear production at the end of the

283

study (day 33) compared to before the start of weekly administrations (day 5) (p < 0.05), but it

284

was only able to restore up to about 50% of the initial tear production rate (day 1). These

285

findings were also corroborated by the corneal fluorescein staining analysis. All groups

286

(excluding the Healthy control group) showed a similar dryness on the eye: compared to the

287

Healthy group, all groups showed a relatively high amount of fluorescein staining on the corneal



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Page 14 of 33

288

surface due to the ocular surface damage inflicted by the dryness induced by the treatment. Only

289

the NP-CsA 0.005-0.01% group showed an improvement by the end of the study compared to its

290

earlier measurements in terms of corneal fluorescein staining, whereas the NP-CsA 0.025%

291

group showed improvements up to week 2 (day 19), but showed a worsening effect from day 19

292

until day 33.

293

Histopathology analysis. The ocular tissues of the mice in the Healthy group showed all the

294

signs of healthy eyes. The conjunctival epithelium displayed surface epithelium cells with

295

normal morphology and architecture along with abundant goblet cells which indicated the

296

presence of adequate conditions for production and formation of a stable tear film layer on the

297

surface of the ocular mucous membrane (Figure 7, A). The lamina propria were composed of

298

loose connective tissues with occasional lymphocytes, and showed no evidence of acute or

299

chronic inflammation. By contrast, in the DES groups treated with both the Saline and Blank

300

NPs showed conjunctival epithelium and lamina propria changes consistent with disease (Figure

301

7, B and C). There were mild to moderate levels of mixed inflammatory infiltrates composed of

302

lymphocytes and occasional polymorphonuclears leucocytes, indicated by the red arrows in the

303

figure. The epithelium was thinned out and showed marked reduction, or in some cases,

304

complete absence of goblet cells. The mice treated with NP-CsA 0.005-0.01% showed

305

conjunctiva with no signs of inflammatory infiltrates (Figure 7, D), displaying morphological

306

features similar to those seen in the Healthy mice group. Moreover, the surface epithelium

307

demonstrated partial to complete recovery of the goblet cells. In terms of the number of goblet

308

cells, the majority of the surface epithelium of the eye samples in this group closely resembled

309

the Healthy group. Similarly, the mice treated with higher dose of CsA loaded NPs (NP-CsA

310

0.025%) showed no signs of inflammatory infiltrates (Figure 7, E). However, the ocular


Page 15 of 33

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311

specimens in this group demonstrated a significantly reduced amount of goblet cells on the

312

surface, and in some cases, complete lack of goblet cells. The mice treated with the commercial

313

form of CsA, RESTASIS®, similarly showed no signs of inflammation, but similar to the NP-

314

CsA 0.025% group, the surface epithelium remained thinned and was completely deprived of

315

goblet cells (Figure 7, F).

316 317

DISCUSSION

318

Mucoadhesive NPs were developed to enhance the delivery of ocular therapeutics to the

319

anterior segment of the eye. PLA-b-Dex NPs, with PBA grafted onto the dextran surface for

320

mucosal targeting, previously showed enhanced in vitro mucoadhesion and sustained release of

321

CsA from the NPs for up to 5 days.33 In a subsequent study, we demonstrated that the CsA

322

loaded NPs showed compatibility on rabbit eyes and efficacy in treating experimental DES in

323

mice using once a week administration.38 Because the previous studies were limited to a short

324

term treatment period of 1 week, the aim of the current study was to evaluate long term treatment

325

effects administering CsA loaded NPs once a week. The experimental DES was induced in mice

326

using the combination of transdermal application of anticholinergic agents, scopolamine, and the

327

exposure to desiccating environments, as reported in previous studies.41, 43 From these studies,

328

we hypothesized that the mucoadhesive and sustained release properties of the NPs prolong the

329

retention of CsA, thus allowing for the treatment of dry eye with significantly reduced dosage of

330

CsA in the formulation.

331

Although we previously demonstrated the in vitro mucoadhesion properties of PBA modified

332

NPs, in this study, we further evaluated the covalent interaction between the PBAs on the surface

333

of the NPs with SA molecules, which are abundantly found on the terminal oligosaccharide side



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Page 16 of 33

334

chains of the glycoproteins of mucin.44 The interaction between PBA and SA was quantified by

335

evaluating the extent of the fluorescence quenching of the PBA molecules as a result of the

336

covalent complexation formed between PBA and SA through photoinduced electron transfer.45

337

Using the relative fluorescence intensity data, we also obtained a binding constant value (KSV =

338

(1.38 ± 0.3) × 104 M-1) much higher than the literature values of PBA modified particles.40 This

339

is most likely due to the large number of PBA’s modified onto the backbone of dextran of our

340

NPs (12.2 mol/mol% PBA on dextran monomers) compared to PBA modified PEGylated

341

particles, which can only have one PBA functional group per PEG chain. From this study, not

342

only did we confirm the in vitro mucoadhesion property that we demonstrated previously, but we

343

also demonstrated the covalent interaction between the PBA’s on the NP surface and the SA.

344

In this study, we demonstrated prolonged retentions of both the PBA modified NP shells and

345

the model drug delivered through PBA modified NPs using fluorescence imaging using in vivo

346

studies. We first showed that the shell material of the nanoparticles, PBA modified dextran,

347

showed improved retention compared to unmodified dextran for up to 6 hrs based on qualitative

348

observation, demonstrating the mucoadhesive properties of PBA molecules. We also

349

demonstrated the prolonged retention of the model drug, ICG, when delivered with PBA

350

modified NPs in vivo compared against an aqueous solution ICG encapsulated in NPs showed

351

increased retention on the surface of rabbit eyes for up to 24 hrs using quantitative analysis

352

(Figure 4). Considering the rapid clearance of Restasis® from the ocular surface within the first

353

20 minutes of eye drop administration,46 these data demonstrate that PBA modified NPs may

354

significantly prolong the ocular retention of loaded therapeutics by attaching to the ocular

355

mucous membrane. Previous studies demonstrated improved retention of CsA by encapsulating

356

them in non-PBA NPs but the CsA concentration in the lachrymal fluid quickly reached baseline


Page 17 of 33

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357

levels after the first few hours.5, 47 This study further corroborates the previous in vitro data

358

showing mucoadhesive properties of the PBA modified NPs due to covalent linkage to mucin.33

359

We next studied how prolonged ocular retention of drugs could improve the long term

360

treatment effect of CsA-loaded NPs on dry eye conditions induced in mice. The dry eye

361

induction was confirmed by the drastic drop in tear production rate within 4 days (Figure 5), as

362

well as the increased amount of fluorescein staining on the corneal surface (day 5) compared to

363

the Healthy control group (Figure 6). From both tear production measurements and fluorescein

364

staining analysis, most of the mice in the groups after dry eye induction (excluding the Healthy

365

group) maintained the dry eye conditions throughout the study (from day 5 to day 33) without

366

much improvement in these clinical parameters. The only groups that showed statistically

367

significant improvements between day 5 and day 33 were the Saline and NP-CsA 0.025% groups

368

(p < 0.05), but comparably, the increased tear production is only at 50% of the initial tear

369

production rate. This phenomenon was also observed in our previous short term treatment

370

study.39 Although the tear production improved for NP-CsA 0.025%, the corneal fluorescein

371

staining only improved up to day 19 and gradually worsened toward the end of the study,

372

implying distinct ocular surface damage. We found that although the tear production

373

measurements and corneal fluorescein clearances provided some insights to the symptoms of the

374

dry eye, the treatment effects were mostly masked by the harsh dry eye induction procedures

375

(scopolamine and desiccating environment) maintained throughout the study. To better

376

understand the treatment effects of the immunosuppressant agent, CsA, closer examination of the

377

ocular surface was conducted using histopathology, to identify signs of inflammation and ocular

378

surface integrity. The ocular tissues of the Saline and Blank NPs group showed that these

379

formulations were unable to treat the symptoms of DES, demonstrating pronounced



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Page 18 of 33

380

inflammation and a complete lack of goblet cells on the ocular surface (Figure 7). It is possible

381

that Blank NPs group may further induce inflammation due to the degradation of the PLA

382

chains. However, in our previous study, we showed that the NPs themselves did not cause any

383

inflammation after weekly dosing of up to 12 weeks.39 It is likely that the timeframe for NP

384

clearance with the mucous membrane is shorter than that of hydrolytic degradation of the PLA

385

polymers.48, 49 All three formulations containing CsA (NP-CsA 0.005-0.01%, NP-CsA 0.025%,

386

and Restasis®) showed elimination of inflammatory infiltrates, which is the main function of

387

CsA. However, only the NP-CsA 0.005-0.01% group showed recovery of the ocular surface in

388

addition to the lack of inflammatory infiltrates. In our previous study, we observed the

389

elimination of inflammation and full recovery of the ocular surface with NP-CsA 0.025% group

390

for the 1 week treatment study.39

391

In this study, after 4 weeks of treatment, the NP-CsA 0.005-0.01% showed both the

392

elimination of inflammation and the recovery of the ocular surface while NP-CsA 0.025% only

393

showed elimination of inflammation without the full ocular surface recovery. We speculate that

394

NP-CsA 0.025% group likely had sufficient CsA to treat DES in 1 week, but when this dosage

395

was repeated for 4 weeks, the prolonged exposure to this dose may have slowed down or

396

prevented the recovery of the ocular surface. The long term toxicity of CsA on the ocular surface

397

has not been well documented in the past,50 likely due to rapid ocular clearance of CsA using

398

conventional formulations. We hope to further investigate the long term effect of CsA on ocular

399

surface as a result of mucoadhesion of the drug carriers, especially in terms of recovering the

400

ocular surface tissues. The mice treated with Restasis® also showed similar effects, likely due to

401

the frequent exposure (thrice daily) to the high concentration CsA in the precorneal tear fluid.

402

Therefore, we believe that it is necessary to lower the dosage of the CsA in the mucoadhesive


Page 19 of 33

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403

NPs even further, from 5% of Restasis® (NP-CsA 0.025%) to 1 to 2% (NP-CsA 0.005-0.01%),

404

for long term treatment of DES to facilitate the ocular surface recovery without compromising

405

the therapeutic efficacy.

406

CONCLUSION

407

PLA-b-Dex NPs surface grafted with PBA ligands were formulated as a mucoadhesive NP eye

408

drop delivery platform. The mucoadhesive nature of the NPs were further demonstrated by

409

showing enhanced ocular retention of the fluorescent dye, ICG, delivered using the NPs

410

compared to ICG delivered without the NPs. Both once a week administration of NPs (NP-CsA

411

0.025%) and three times daily administration of Restasis® to dry eye induced mice showed

412

elimination of inflammatory infiltrates but failed to fully recover the ocular surface. However,

413

once a week dosing of NPs with an even lower dosage (NP-CsA 0.005-0.01%) showed efficacy

414

in terms of both the elimination of inflammatory infiltrates and the full recovery of the ocular

415

surface, as determined by histopathology. The current study provides promising results for the

416

potential application of PBA-grafted mucoadhesive NPs to dramatically prolong the dosing

417

interval, increase compliance and improve the efficacy of CsA and other ocular therapeutics for

418

treating anterior eye diseases.

419

ASSOCIATED CONTENT

420

Supporting Information

421

In vitro release of CsA from PBA modified NPs in simulated tear fluid. This material is

422

available free of charge via the Internet at http://pubs.acs.org.

423

AUTHOR INFORMATION

424

Corresponding Author



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

425

*E-mail: [email protected]. QNC 4602 – University of Waterloo. 200 University Ave

426

West. Waterloo, ON. N2L 3G1.

427

ACKNOWLEDGEMENT

428

This work was financially supported by 20/20 Natural Sciences and Engineering Research

429 430

Council - Ophthalmic Materials Network and AmorChem Holdings Inc. REFERENCES

431

(1). Lemp MA, Baudouin C, Baum J, et al. The definition and classification of dry eye disease:

432

Report of the definition and classification subcommittee of the international dry eye WorkShop

433

(2007). Ocul Surf. 2007;5:75-92.

434

(2). Sall K, Stevenson OD, Mundorf TK, Reis BL, CsA Phase 3 Study Grp. Two multicenter,

435

randomized studies of the efficacy and safety of cyclosporine ophthalmic emulsion in moderate

436

to severe dry eye disease. Ophthalmol. 2000;107:631-639.

437

(3). Gaudana R, Jwala J, Boddu SHS, Mitra AK. Recent perspectives in ocular drug delivery.

438

Pharm Res. 2009;26:1197-1216.

439

(4). Barber LD, Pflugfelder SC, Tauber J, Foulks GN. Phase III safety evaluation of

440

cyclosporine 0.1% ophthalmic emulsion administered twice daily to dry eye disease patients for

441

up to 3 years. Ophthalmology 2005;112:1790-1794.

442

(5). Yavuz B, Pehlivan SB, Unlu N. An overview on dry eye treatment: Approaches for

443

cyclosporin A delivery. Scientific World Journal 2012:194848.

444

(6). Mantelli F, Tranchina L, Lambiase A, Bonini S. Ocular surface damage by ophthalmic

445

compounds. Curr Opin Allergy Clin Immunol. 2011;11:464-470.


Page 20 of 33

Page 21 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


446

(7). Iester M, Telani S, Frezzotti P, et al. Ocular surface changes in glaucomatous patients

447

treated with and without preservatives beta-blockers. J Ocul Pharmacol Ther. 2014;30:476-481.

448

(8). Jee D, Park SH, Kim MS, Kim EC. Antioxidant and inflammatory cytokine in tears of

449

patients with dry eye syndrome treated with preservative-free versus preserved eye drops. Invest

450

Ophthalmol Vis Sci. 2014;55:5081-9.

451

(9). Aydin Kurna S, Acikgoz S, Altun A, Ozbay N, Sengor T, Olcaysu OO. The effects of

452

topical antiglaucoma drugs as monotherapy on the ocular surface: A prospective study. J

453

Ophthalmol. 2014;2014.

454

(10). Liu S, Jones L, Gu FX. Nanomaterials for ocular drug delivery. Macromol Biosci.

455

2012;12:608-620.

456

(11). Wadhwa S, Paliwal R, Paliwal SR, Vyas SP. Nanocarriers in ocular drug delivery: An

457

update review. Curr Pharm Des. 2009;15:2724-2750.

458

(12). Sahoo SK, Diinawaz F, Krishnakumar S. Nanotechnology in ocular drug delivery. Drug

459

Discov Today. 2008;13:144-151.

460

(13). Diebold Y, Calonge M. Applications of nanoparticles in ophthalmology. Prog Retin Eye

461

Res. 2010;29:596-609.

462

(14). Cho HK, Cheong IW, Lee JM, Kim JH. Polymeric nanoparticles, micelles and

463

polymersomes from amphiphilic block copolymer. Korean J Chem Eng. 2010;27:731-740.

464

(15). Subbiah R, Veerapandian M, Yun KS. Nanoparticles: Functionalization and multifunctional

465

applications in biomedical sciences. Curr Med Chem. 2010;17:4559-4577.

466

(16). Shinde UA, Shete JN, Nair HA, Singh KH. Eudragit RL100 based microspheres for ocular

467

administration of azelastine hydrochloride. J Microencapsul. 2012;29:511-519.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

468

(17). Kimura H, Ogura Y. Biodegradable polymers for ocular drug delivery. Ophthalmologica.

469

2001;215:143-155.

470

(18). Gavini E, Chetoni P, Cossu M, Alvarez MG, Saettone MF, Giunchedi P. PLGA

471

microspheres for the ocular delivery of a peptide drug, vancomycin using emulsification/spray-

472

drying as the preparation method: In vitro/in vivo studies. Eur J Pharm Biopharm. 2004;57:207-

473

212.

474

(19). Yoncheva K, Vandervoort J, Ludwig A. Development of mucoadhesive poly(lactide-co-

475

glycolide) nanoparticles for ocular application. Pharm Dev Technol. 2011;16:29-35.

476

(20). Gupta H, Aqil M, Khar RK, Ali A, Bhatnagar A, Mittal G. Sparfloxacin-loaded PLGA

477

nanoparticles for sustained ocular drug delivery. Nanomed-Nanotechnol. 2010;6:324-333.

478

(21). Aliabadi HM, Mahmud A, Sharifabadi AD, Lavasanifar A. Micelles of methoxy

479

poly(ethylene oxide)-b-poly(epsilon-caprolactone) as vehicles for the solubilization and

480

controlled delivery of cyclosporine A. J Control Release. 2005;104:301-311.

481

(22). Velluto D, Demurtas D, Hubbell JA. PEG-b-PPS diblock copolymer aggregates for

482

hydrophobic drug solubilization and release: Cyclosporin A as an example. Mol Pharm.

483

2008;5:632-642.

484

(23). Mondon K, Zeisser-Labouebe M, Gurny R, Moeller M. Novel cyclosporin A formulations

485

using MPEG-hexyl-substituted polylactide micelles: A suitability study. Eur J Pharm Biopharm.

486

2011;77:56-65.

487

(24). Bazile D, Prudhomme C, Bassoullet MT, Marlard M, Spenlehauer G, Veillard M. Stealth

488

me.peg-pla nanoparticles avoid uptake by the mononuclear phagocytes system. J Pharm Sci.

489

1995;84:493-498.


Page 22 of 33

Page 23 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


490

(25). Dhar S, Gu FX, Langer R, Farokhzad OC, Lippard SJ. Targeted delivery of cisplatin to

491

prostate cancer cells by aptamer functionalized pt(IV) prodrug-PLGA-PEG nanoparticles. Proc

492

Natl Acad Sci U.S.A. 2008;105:17356-17361.

493

(26). Dong Y, Feng S. In vitro and in vivo evaluation of methoxy polyethylene glycol-

494

polylactide (MPEG-PLA) nanoparticles for small-molecule drug chemotherapy. Biomaterials.

495

2007;28:4154-4160.

496

(27). Esmaeili F, Ghahremani MH, Ostad SN, et al. Folate-receptor-targeted delivery of

497

docetaxel nanoparticles prepared by PLGA-PEG-folate conjugate. J Drug Target. 2008;16:415-

498

423.

499

(28). Gao Y, Sun Y, Ren F, Gao S. PLGA-PEG-PLGA hydrogel for ocular drug delivery of

500

dexamethasone acetate. Drug Dev Ind Pharm. 2010;36:1131-1138.

501

(29). Vega E, Egea MA, Calpena AC, Espina M, Garcia ML. Role of hydroxypropyl-beta-

502

cyclodextrin on freeze-dried and gamma-irradiated PLGA and PLGA-PEG diblock copolymer

503

nanospheres for ophthalmic flurbiprofen delivery. Int J Nanomedicine. 2012;7:1357-1371.

504

(30). Yang J, Yan J, Zhou Z, Amsden BG. Dithiol-PEG-PDLLA micelles: Preparation and

505

evaluation as potential topical ocular delivery vehicle. Biomacromolecules. 2014:1346-1354.

506

(31). Verma MS, Liu S, Chen YY, Meerasa A, Gu FX. Size-tunable nanoparticles composed of

507

dextran-b-poly(D,L-lactide) for drug delivery applications. Nano Res. 2012;5:49-61.

508

(32). Goodwin AP, Tabakman SM, Welsher K, Sherlock SP, Prencipe G, Dai H. Phospholipid-

509

dextran with a single coupling point: A useful amphiphile for functionalization of nanomaterials.

510

J Am Chem Soc. 2009;131:289-296.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

511

(33). Liu S, Jones L, Gu FX. Development of mucoadhesive drug delivery system using

512

phenylboronic acid functionalized poly(D,L-lactide)-b-dextran nanoparticles. Macromol Biosci.

513

2012;12:1622-1626.

514

(34). Li N, Zhuang C, Wang M, Sui C, Pan W. Low molecular weight chitosan-coated liposomes

515

for ocular drug delivery: In vitro and in vivo studies. Drug Deliv. 2012;19:28-35.

516

(35). Mahmoud AA, El-Feky GS, Kamel R, Awad GEA. Chitosan/sulfobutylether-beta-

517

cyclodextrin nanoparticles as a potential approach for ocular drug delivery. Int J Pharm.

518

2011;413:229-236.

519

(36). Abdelbary G. Ocular ciprofloxacin hydrochloride mucoadhesive chitosan-coated

520

liposomes. Pharm Dev Technol. 2011;16:44-56.

521

(37). Li N, Zhuang C, Wang M, Sun X, Nie S, Pan W. Liposome coated with low molecular

522

weight chitosan and its potential use in ocular drug delivery. Int J Pharm. 2009;379:131-138.

523

(38). De Campos AM, Sanchez A, Alonso MJ. Chitosan nanoparticles: A new vehicle for the

524

improvement of the delivery of drugs to the ocular surface. application to cyclosporin A. Int J

525

Pharm. 2001;224:159-168.

526

(39). Liu S, Chang CN, Verma M, et al. Phenylboronic acid modified mucoadhesive nanoparticle

527

drug carriers facilitate weekly treatment of experimentally-induced dry eye syndrome. Nano Res.

528

2014.

529

(40). Deshayes S, Cabral H, Ishii T, et al. Phenylboronic acid-installed polymeric micelles for

530

targeting sialylated epitopes in solid tumors. J.Am.Chem.Soc. 2013;135:15501-15507.

531

(41). Dursun D, Wang M, Monroy D, et al. A mouse model of keratoconjunctivitis sicca. Invest

532

Ophthalmol Vis Sci. 2002;43:632-638.


Page 24 of 33

Page 25 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


533

(42). Okanobo A, Chauhan SK, Dastjerdi MH, Kodati S, Dana R. Efficacy of topical blockade of

534

interleukin-1 in experimental dry eye disease. Am.J.Ophthalmol. 2012;154:63-71.

535

(43). Oh HJ, Li Z, Park S, Yoon KC. Effect of hypotonic 0.18% sodium hyaluronate eyedrops on

536

inflammation of the ocular surface in experimental dry eye. J Ocul Pharmacol Ther.

537

2014;30:533-42.

538

(44). Khutoryanskiy VV. Advances in mucoadhesion and mucoadhesive polymers. Macromol

539

Biosci. 2011;11:748-764.

540

(45). JAMES T, SANDANAYAKE K, SHINKAI S. Chiral discrimination of monosaccharides

541

using a fluorescent molecular sensor. Nature 1995;374:345-347.

542

(46). Di Tommaso C, Valamanesh F, Miller F, et al. A novel cyclosporin A aqueous formulation

543

for dry eye treatment: In vitro and in vivo evaluation. Invest.Ophthalmol.Vis.Sci. 2012;53:2292-

544

2299.

545

(47). Cholkar K, Patel SP, Vadlapudi AD, Mitra AK. Novel strategies for anterior segment

546

ocular drug delivery. Journal of Ocular Pharmacology and Therapeutics 2013;29:106-123.

547

(48). Tracy MA, Ward KL, Firouzabadian L, et al. Factors affecting the degradation rate of

548

poly(lactide-co-glycolide) microspheres in vivo and in vitro. Biomaterials 1999;20:1057-1062.

549

(49). Kim K, Yu M, Zong XH, et al. Control of degradation rate and hydrophilicity in

550

electrospun non-woven poly(D,L-lactide) nanofiber scaffolds for biomedical applications.

551

Biomaterials 2003;24:4977-4985.

552

(50). Lallemand F, Felt-Baeyens O, Besseghir K, Behar-Cohen F, Gurny R. Cyclosporine A

553

delivery to the eye: A pharmaceutical challenge. European Journal of Pharmaceutics and

554

Biopharmaceutics 2003;56:307-318.

555



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Page 26 of 33

556 557 558 559 560 561 562

Tables

563

Table 1. 6 different groups applied to the mice for treatment of experimental dry eye. Treatment group

Dry eye induced

Admin. volume (µl)

NP (µg)

CsA (µg)

Admin. frequency

Healthy

No

0

0

0

Saline

Yes

7

0

0

x1/week

Blank NP

Yes

7

4

0

x1/week

NP-CsA 0.0050.01%

Yes

7

4

0.3/0.6*

x1/week

NP-CsA 0.025%

Yes

7

4

1.5

x1/week

Restasis®

Yes

3

0

1.5

x3/day

564 565

*NP-CsA 0.005-0.01% was administered with 0.3 µg of CsA for the first two weeks, and with 0.6 µg of CsA for the subsequent two weeks.

566 567

Table 2. Nanoparticle diameters measured using dynamic light scattering (DLS). MSD* diameter (nm) Polydispersity Blank NPs

25.4 ± 0.6

0.240

NP-CsA 0.005-0.01%

26.9 ± 1.1

0.317


Page 27 of 33

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NP-CsA 0.025% 568

29.1 ± 0.3

0.199

*MSD: Multimode-size distribution.

569 570 571 572 573 574 575 576 577

FIGURES

578 579

Figure 1. In vitro interaction between PBA-modified NPs and sialic acid (SA). a) Emission

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spectra of PBA-modified NPs (50 µg/ml) in various concentrations of SA (0 to 0.32 mM) at

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room temperature, λex = 298 nm. b) Relative fluorescence as a function of SA concentrations for

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PBA-modified. I0 and I represent the fluorescence intensity in the absence and presence of SA

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respectively. Data were fit according to Stern-Volmer equation.

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Figure 2. Images of NZW rabbit eyes treated with cyanine labelled dextran (Dex-cyanine – PBA)

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and PBA-dextran (Dex-cyanine + PBA) obtained with confocal Scanning Laser Ophthalmoscope

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(cSLO), λex = 795 nm and λem = 810 nm.


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Figure 3. Images of NZW rabbit eyes treated with Free ICG, NP-ICG (- PBA), and NP-ICG (+

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PBA) obtained with cSLO, λex = 795 nm and λem = 810 nm.



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Figure 4. ImageJ analysis of the fluorescence images taken from rabbit eyes treated with Free

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ICG, NP-ICG (- PBA) and NP-ICG (+ PBA) (n = 3; mean ± s.e.m.). I: fluorescence brightness

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measured using ImageJ software; I0: initial fluorescence brightness (at 0 hr). Student t-test

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between NP-ICG (+ PBA) and NP-ICG (- PBA): ** = p < 0.05 and * = p < 0.1.

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Figure 5. Tear production measurement of mice with different treatment groups. The tear

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volumes (T) were normalized with respect to their initial tear volume (T0). The arrows represent

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the weekly dosing regimen of Saline, Blank NPs, NP-CsA 0.005-0.01% and NP-CsA 0.025%

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groups.


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Figure 6. Corneal fluorescein staining analysis of mice with different treatment groups. The

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arrows represent the weekly dosing regimen of Saline, Blank NPs, NP-CsA 0.005-0.01% and

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NP-CsA 0.025% groups. Note that the analysis does not take into account the fluorescein on the

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lid margins of the eyes.

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Figure 7. Histopathology analysis of ocular tissues of mice with different treatments: A) Healthy,

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B) Saline, C) Blank NPs, D) NP-CsA 0.005-0.01%, E) NP-CsA 0.025%, and F) Restasis®. The

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scale bars are 100 µm in length. The red arrows represent some of the inflammatory infiltrates

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such as lymphocytes, polymorphonuclears and eosinophils observed.


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TABLE OF CONTENTS. Nanoparticle eye drop delivery platform is formed by the selfassembly of poly(D,L-lactide)-b-dextran-g-phenylboronic acid (PLA-b-Dex-g-PBA) block copolymers and Cyclosporine A, a dry eye therapeutic drug. The mucoadhesive targeting of PBA on the nanoparticle surface and the controlled drug release properties of the nanoparticles facilitate the treatment of dry eye using once a week dosing of the nanoparticle formulation.

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Prolonged Ocular Retention of Mucoadhesive ... - ACS Publications

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