Research paper
Polymeric triamcinolone acetonide nanoparticles as a new alternative in the treatment of uveitis: In vitro and in vivo studies

https://doi.org/10.1016/j.ejpb.2012.12.010Get rights and content

Abstract

The purpose of this work was to improve the efficacy of triamcinolone acetonide (TA) in the treatment of endotoxin-induced uveitis (EIU) using a polymeric nanoparticulate drug delivery system. Poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles were prepared using a modified emulsification/solvent diffusion method. Processing factors affecting loading and size were also studied. After physicochemical studies including in vitro release, X-ray powder diffraction, differential scanning calorimetry, and scanning electron microscopy, in vivo studies were conducted using nanoparticles sized 195 nm with 3.16% drug loading. Inflammatory factors such as flare, cell, and fibrin were studied in rabbit’s eye over 96 h period, using laser flare meter and slit lamp examination. Inflammatory mediators such as NO, PGE2, cell, and protein were measured quantitatively 36 h after intravitreal injection of endotoxin in aqueous humor, and the therapeutic effects were compared in different groups. Results indicated statistically significant differences between the effect of nanoparticles in the treatment of EIU compared to microparticles of TA and prednisolone acetate (PA). There were no significant differences between the effects of TA injection and TA nanoparticles. In conclusion, sustain release biodegradable TA nanoparticles are potential new topical treatment options which can provide better patient compliance.

Graphical abstract

PLGA polymer was used to prepare polymeric triamcinolone acetonide (TA) nanoparticles. After physicochemical studies, two parallel in vivo studies were conducted on rabbit eyes with endotoxin-induced uveitis using PLGA–TA nanoparticles. Results showed that PLGA–TA nanoparticles could be considered as a potential alternative to common treatments.

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Introduction

Uveitis is considered to be one of the leading causes of blindness that may be associated with systemic inflammation. It causes the inflammation of uvea and neuro-retina which may result in serious loss of vision and morbidity [1], [2]. As with many other ocular conditions such as macular edema, age related macular degeneration, and uveitic cystoid macular edema, uveitis, is treated with subtenon, subconjunctival, or intravitreal injection of TA which is a water-insoluble corticosteroid [3], [4]. Each of these three injection methods for drug delivery is associated with certain complications. For instance, intravitreal injections can accelerate cataract formation and cause hemorrhage, retinal detachment, endophthalmitis and increased intraocular pressure [5], [6]. In case of subtenon and subconjunctival injections, the drug does not penetrate the vitreous enough to achieve therapeutic concentrations equal to intravitreal injections, and thus, higher doses of TA are used which can lead to a severe increase in the intraocular pressure as a side effect [7].

Issues of ocular drug delivery include not only biologic barriers of the eye such as corneal tight junctions, but also exclusive ocular barriers such as baseline and reflex lacrimation, blinking, and nasolacrimal drainage which are not seen with drug delivery to other organs. All these factors reduce the half-life of drug presence on the ocular surface and the pre-corneal area down to 1–3 min after instillation [6], [8].

In the past 20 years, great effort has been made to overcome the challenges in ophthalmic drug delivery, and all proposed methods have advantages as well as drawbacks which include discomfort, blurred vision, foreign body sensation, and lid adhesion which are associated with increased viscosity of drug carrier and inserts [8], [9], endothelial damage due to implant migration with ocular implants [10], ptosis and fibrosis due to periocular injections [11], increased intraocular pressure, hemorrhage and cataracts which are associated with intravitreal injections [12] and visual impairment due to the use of microparticles for the posterior segment [13].

Many comprehensive investigations in recent years indicate that the future of ocular drug delivery belongs to colloidal systems (hydrogels, microparticles, liposomes, nanoparticles), especially colloidal systems at nano-scale. The advantages of nanosuspensions include increased solubility, surface area and dissolution rate of water-insoluble drugs [14], [15] as well as increased ophthalmic bioavailability, bio-adhesion and elimination half-life in tears [16], [17], [18].

The application of polymeric nanoparticles (NPs) containing drugs, such as PLGA, poly-caprolactone and poly-alkylcyanoacrylate increase the corneal uptake [19], intraocular penetration [20], [21], and conjunctival uptake of the drug [22] compared to solutions and microparticulate preparations. Overall, nanoparticles increase drug presence in the ocular and target tissue while patient compliance is comparable to topical solutions.

Considering the abovementioned advantages of ocular drug delivery using polymeric NPs, the complications of ocular injections and the fact that the concentration of NPs in inflamed eyes are many times higher than that in normal eyes [23], so nanoparticulate drug delivery systems may be regarded as the next treatment option for eye disease, especially for ocular inflammations.

The purpose of our investigation was to use the PLGA polymer to prepare NPs of TA, evaluate preparation factors in terms of their effects on NPs characteristics, and also analyze the physicochemical properties. As an in vivo study, anti-inflammatory effects of topically administered PLGA–TA NPs were compared with that of PA microsuspension eye drops, TA microsuspension eye drops, and TA injection in an animal model of uveitis.

PLGA is a biodegradable polymer which has been widely used in nano-drug delivery research [24], [25], has FDA approval for use in drug preparation, increases sustain release time and protects drugs from enzymatic degradation [26], [27].

In this study, animal models of uveitis were induced by administering intraocular injection of lipopolysaccharide (LPS), a part of the outer membrane of Gram-negative bacteria. This acute anterior uveitis is known as EIU. LPS may directly activate the vascular endothelium, macrophages and other cells subsequently lead to cellular infiltration and protein (flare) extravasation in the uveal tract [28], [29]. Secretion of various inflammatory mediators, such as prostaglandin E2 (PGE2) and nitric oxide (NO) in the aqueous humor of the inflamed eye, is common in EIU [30].

Clinical marks of ocular inflammation are usually detected subjectively with the use of a slit lamp biomicroscopy system and graded by an ophthalmologist using standardized scoring systems based on inflammation elements in eye [2], [31]. As an example the amount of protein, cell and inflammatory mediators such as PGE2 and NO in the aqueous humor have been investigated to assess the efficiency of different herbal medicines in the treatment of EIU [32]. In this study, Kowa laser flare meter (LFM), the most objective, qualitative, accurate and non-invasive method for monitoring uveitis was used, which can also help compare the effects of different drugs on uveitis and measure aqueous humor flare. The LFM determines aqueous flare by measuring light scattering of a laser beam in the anterior chamber [33], [34], [35].

Section snippets

Materials

TA was purchased from Crystal Pharma (Spain). PLGA (50:50 d,l-lactide:glycolide) Resomer 502H MW 12,000 Da was obtained from Boehringer–Ingelheim (Germany). Poly vinyl alcohol (PVA 88% hydrolyzed, MW 20,000–30,000) was purchased from Acros (Belgium). Acetone, ethanol, dichloromethane (analytical grade), and acetonitrile (high-performance liquid chromatography (HPLC) grade) were purchased from Duksan (South Korea). Ethylene diamine tetra acetic acid (EDTA) was obtained from Merck (Germany).

Preparation of nanoparticles

To

Nanoparticles formulation

Fig. 1 shows the SEM micrograph of PLGA–TA NPs prepared using modified emulsification/solvent diffusion method. Previous works have shown that NPs of about 200 nm can be prepared for lipophilic drugs with the same method [27], [39]. One of the challenges of this method is the formation of drug crystals, while NPs are forming. Gómez-Gaete et al. [36] demonstrated the presence of dexamethasone crystals with PLGA–dexamethasone NPs formation which affects the entrapment efficiency and results in a

Conclusion

Based on our observations, when preparing NPs, TA can be well encapsulated in PLGA polymer. Successive filtration can eliminate drug crystals that form during the preparation process which decreases the burst effect and prevents overestimation of entrapment efficiency. The NPs prepared with PLGA polymer showed a controlled release profile with drug/polymer ratio of 1/10. This was associated with a 30% burst effect which seems an ideal drug delivery option for the treatment of uveitis and other

Acknowledgments

Authors would like to thank Dr. Shiva Mehravaran and Dr. Mirgholamreza Mahbod for their technical assistance in animal studies and Valuable advices and technical support from Dr. Mohammad Reza Khoshayand for statistical analysis. This work was financially supported by a grant from Tehran University of Medical Sciences.

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