Elsevier

The Ocular Surface

Volume 16, Issue 4, October 2018, Pages 415-423
The Ocular Surface

Original Research
Subconjunctival dendrimer-drug therapy for the treatment of dry eye in a rabbit model of induced autoimmune dacryoadenitis

https://doi.org/10.1016/j.jtos.2018.05.004Get rights and content

Abstract

Purpose

To investigate the efficacy of a single subconjunctival injection of dendrimer-dexamethasone conjugate in a rabbit model of induced autoimmune dacryoadenitis (AID).

Methods

Dendrimer biodistribution after subconjunctival injection in AID animals was evaluated using Cy5-labelled dendrimer (D-Cy5) and confocal microscopy. Diseased animals were treated with free dexamethasone (Free-Dex), dendrimer-dexamethasone (D-Dex), or saline via a single subconjunctival injection. The efficacy was evaluated using various clinical evaluations, such as Schirmer's test, tear breakup time (TBUT), and fluorescein and rose Bengal staining. Histopathology was evaluated by H&E staining and immunostaining. Levels of inflammatory cytokines and aquaporin proteins in the LGs were determined by real-time PCR.

Results

Subconjunctivally administered dendrimers selectively localized in the inflamed LGs, and were taken up by the infiltrating cells. At two weeks post single dose-treatment, the D-Dex group showed improved clinical evaluations. No significant changes were observed in other groups. H&E staining demonstrated less inflammatory cell infiltration and fewer atrophic acini in D-Dex group, compared to those treated with saline or Free-Dex. Immunohistochemistry demonstrated that the intensity of CD-18 (+) and RTLA (+) was weaker in LGs in the D-Dex group than in other treatment groups. Pro-inflammatory gene expression levels of MMP9, IL6, IL8, and TNFα were significantly decreased in the D-Dex group compared to the Free-Dex and saline group.

Conclusions

The dendrimer exhibits pathology-dependent biodistribution in the inflamed LGs. Subconjunctivally administered D-Dex suppressed LG inflammation, leading to partial recovery of LG function with clinical improvement in induced AID. Sjögren's patients may benefit from this targeted nanomedicine approach.

Introduction

Dry eye disease (DED) is a widely prevalent eye disorder affecting 46 million Americans, especially the elderly population [1,2]. It is a multifactorial disease which causes various degrees of dryness of the eyes and discomfort, blurred vision, ocular surface damage, and even functional blindness [3]. One of the main causes of DED is Sjögren's syndrome, an autoimmune disease characterized by focal lymphocytic infiltration and severe dysfunction of the exocrine organs, primarily the lacrimal gland (LG) and salivary gland [[4], [5], [6], [7], [8], [9]]. Although the precise etiology of DED remains unclear, it is believed to be a chronic inflammatory disease especially in Sjögren's syndrome induced DED. A wide range of immunologic, genetic, epigenetic, hormonal, viral, neural, and environmental variables are proposed as being involved in the pathogenesis of Sjögren's syndrome, where lymphocytic infiltration of the LG is associated with exocrine insufficiency, leading to functional quiescence and eventual destruction of the secretory parenchyma [[10], [11], [12]].

Although numerous murine models present with features resembling those of Sjögren's syndrome in humans, none completely represents the pathophysiological characteristics of this condition [9]. An induced rabbit model of chronic autoimmune dacryoadenitis (AID) for studying the pathogenesis and pathophysiology of this disease shares immune features of human Sjögren's syndrome [13,14]. Moreover, the rabbit LG exhibits greater similarity to the microanatomical and immunohistological features of the human LG [15]. As there is a precise onset of disease initiation in this inducible model, this animal model offers a good platform to identify and test candidate therapies, and to further explore underlying mechanisms [8,13,14,16].

Current treatment for DEDs is mainly focused on reducing inflammation and restoring tears [11,17]. Topical anti-inflammatory corticosteroid drops or immunosuppressive drugs such as cyclosporine (CsA) drops are the most common treatments [11,[18], [19], [20]]. Although the aforementioned treatments achieve clinical success in relieving the symptoms and signs of moderate or severe dry eye in Sjögren's syndrome [11,16,20], this treatment requires frequent topical application that often results in corneal irritation, toxicity, and patient non-compliance [21,22]. Furthermore, very little is known about the effects of topical anti-inflammatory drug treatment on the inflammatory cellular response in LGs. Because these topical drugs are not highly permeable, it may be a challenge to deliver and maintain therapeutic concentrations of drugs at the target site in LG. Drug formulations that offer sustained release, cellular targeting, and increased bioavailability may offer new avenues to improve efficacy and reduce side effects in DED treatment [[23], [24], [25]].

Hydroxyl poly(amidoamine) (PAMAM) dendrimers have a well-defined structure, small size (∼3–10 nm), good safety profile, and high water solubility, which makes them excellent candidates for ocular drug and gene delivery [26]. Numerous studies have examined the role of their unique nanoscale architecture on the delivery of therapeutics (drugs and genes) and imaging agents [[26], [27], [28], [29], [30], [31]]. Due to their “neutral” surface charge and nanoscale size, hydroxyl-terminated PAMAM dendrimers are noncytotoxic with minimal nonspecific tissue interactions [23,32,33]. Systemic and intravitreally administered hydroxyl-PAMAM dendrimers are readily cleared intact from off-target organs, but are selectively localized and retained in the areas of inflammation/injury in the brain and retina [[34], [35], [36], [37]]. PAMAM dendrimers target activated macrophages in the injured cornea upon subconjunctival administration, suggesting enhanced delivery and bioavailability of corticosteroids (e.g., dexamethasone) to the very cells responsible for corneal inflammation [23].

Based on the targeted anti-inflammatory activity of the subconjunctival dendrimer-dexamethasone (D-Dex) conjugate in the corneal alkali burn model [23], and its anti-inflammatory activity at 10-fold lower concentration compared to free dexamethasone in lipopolysaccharide-activated murine macrophages [23], we hypothesized that the dendrimer will enhance intracellular delivery and efficacy of drugs on target cells in the LG in a well-established rabbit AID model. In this study, we investigated the efficacy of a single dose of subconjunctivally administered D-Dex in rabbit AID model investigated the dendrimer distribution in the LG, and evaluated the efficacy of dendrimer–drug therapy. We expect the dendrimers may potentially enhance the anti-inflammatory effects of drugs in treatment of severe DED.

Section snippets

Animals and materials

Female adult New Zealand white rabbits, each weighing between 3-4 kg, were obtained from Robinson Services Inc. (Mocksville, NC, USA). The animals were maintained and used in compliance with institutional guidelines and in accordance with the Association for Research in Vision and Ophthalmology Resolution on the Use of Animals in Ophthalmic Research and the U.K. Animals (Scientific Procedures) Act. The research was approved by the Johns Hopkins University animal review board. Clinical

Synthesis of Dendrimer-Cy5 (D-Cy5) and Dendrimer-Dex (D-Dex) conjugates

We used near-IR dye Cy5 for labelling the dendrimer to avoid tissue autofluorescence. 1H NMR and HPLC characterization demonstrated that ∼2 molecules of Cy5 were conjugated to the dendrimer surface and the purity of D-Cy5 was >95%. We previously showed that D-Cy5 is stable in vivo in several animal models [33,41]. A glutaric acid linker was used to conjugate Dex to the dendrimer surface to avoid steric hindrance to conjugation, and to enable drug release through hydrolysis. 1H NMR

Discussion

In this study, we demonstrated that a single subconjunctival injection of D-Dex conjugate has therapeutic effect for DED in an AID model. Current methods of drug introduction to treat Sjögren's syndrome-associated DED are insufficient to successfully treat the disease. Rapid drug elimination from the ocular surface is a major obstacle for topical drug delivery, especially in the case of severe dry eye. Blinking, eyelashes, and reflexive tearing promote rapid removal of eye drops from the eye

Conflicts of interest

The authors declare no conflict of interest and financial or business interest related to this study.

Acknowledgement

Supported in part by an unrestricted grant from Research to Prevent Blindness, New York, NY, to the Wilmer Eye Institute, and NIH/NEI R01 1R01EY025304-01 (RMK).

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