Elsevier

Biomedicine & Pharmacotherapy

Volume 94, October 2017, Pages 402-411
Biomedicine & Pharmacotherapy

Original article
Poly (d, l-lactide-co-glycolide) nanoparticles for sustained release of tacrolimus in rabbit eyes

https://doi.org/10.1016/j.biopha.2017.07.110Get rights and content

Abstract

Tacrolimus (TAC)-loaded Ploy-lactide-co-glycolide nanoparticles (PLGA-NPs) was developed by emulsification-diffusion method for topical ocular delivery in certain ocular conditions where therapeutic level of immunomodulator into eyes is required for sufficient duration. So, we optimized TAC-loaded PLGA-NPs with higher TAC payload. The mean particle-size and its distribution, polydispersity, zeta-potentials, morphology, drug encapsulation and loading capacity of NPs were analyzed. Transcorneal permeation through excised rabbit cornea revealed instant and controlled permeation of TAC from TAC-aqueous suspension (TAC-AqS) and from PLGA-NPs, respectively. Stability study results indicated that there were no significant changes in above characteristics for 1-month storage at 25 °C. The safety of PLAG was established by modified Draize’s test after its topical administration in rabbit eyes. The adopted liquid chromatography-electrospray ionization tandem mass spectrometry was successfully applied for TAC quantification in ocular tissues and aqueous-humor. PLGA-NPs improved corneal, conjunctival and aqueous humor bioavailability of TAC. A considerably higher TAC-concentration from F2 was found in ocular tissues even at 24 h and in aqueous humor till 24 h following its topical ocular administration as compared to TAC-AqS. The PLGA-NPs significantly enhanced ocular bioavailability of TAC than that of aqueous suspension.

Introduction

Topical applications of conventional formulations in the eyes are limited by low and poor ocular availability because of rapid tear-turnover and impermeability of drugs to the cornea due to its defensive mechanism [1]. Cornea is consisting of transparent connective tissues known as stroma, which is protected by epithelia on both the sides. The inner endothelium is a monolayer that outlines the anterior chamber while the outer layer of cornea is made of stratified non keratinized squamous epithelia, which protects the stroma from outer environment by luminal junctions and provides a strong physical barrier against any external materials. The physical barrier is also accompanied by a physicochemical barrier (mucin layer) that defends the entry of any drug or antigen. Moreover, special effects of mechanical washing by tear fluid and wiping of eye lids collectively enhance protective action of the proteins [2]. As a consequence, free drugs in solution form is rapidly eliminated from the ocular surface after instillation in to eyes, thus only 2–5% of the applied dose actually available for the intraocular tissues after corneal and conjunctival permeation and hardly offers high drug availability [3], [4], [5]. So, frequent administration is required to get the therapeutic effects [6], which may cause adverse drug reactions and affect therapeutic and patient compliance. Hence, there is a need of an ideal ocular drug delivery system that would offer a sustained and controlled release of drugs in the eyes. Ploy (d,l-lactide-co-glycolide, 50:50 d,l-lactide:glycolide) nanoparticles (PLGA-NPs) are encouraging ophthalmic delivery systems that are used as sustained and controlled delivery of numerous drugs for ocular disorders [7]. PLGA-NPs are of suitable size for ocular use [8]. Being a biodegradable, biocompatible polymer and its non-toxic degradation byproducts; PLGA has been approved by FDA for ocular use and have strong potential for ocular drug carriers as it resulted very low eye irritation [8], [9]. Polyvinyl alcohol (PVA) and Pluronic-F68 were chosen as stabilizer for the PLGA-NPs development, because PVA [10], [11] and Pluronic F-68 [12], [13] are the surfactants those are free from cytotoxicity.

The immunosuppressants as topical ocular application help in treating the ocular autoimmune diseases, to manage corneal graft rejection, uveitis, ocular pemphigoid, allergic conjunctivitis, dry-eye conditions, keratitis, atopic and vernal keratoconjunctivitis [14], [15], [16], [17], [18], [19], [20] and other ocular inflammatory conditions. PLGA-NPs would provide a sustained ocular delivery of tacrolimus.

Tacrolimus (TAC) is a potent macrolide lactone immunosuppressive agent [21] which was first derived from Streptomyces tsukubaensis. TAC and cyclosporine-A (CsA) are the most common topical immunomodulators having similar mechanism of actions, but TAC is around 10–100 times more potent than CsA [22]. TAC also subdues the immune responses by preventing the release of many other inflammatory cytokines (like, IL-3, IL-4, IL-5, IL-8, Gamma-interferon and tumor necrosis factor-α) [23]. TAC has effectively been used as therapeutic agent in various immune mediated conditions or diseases. TAC was also found to be effective in treating eye conditions such as, delay the incidence of corneal allograft rejection and prolong the allograft survival period [24], ocular inflammation, ocular pemphigoid [25] and uveitis [15].

Topical use of 0.03% TAC eye drops was found effective in severe allergic conjunctivitis and successfully improved ocular surface status and tear stability in dry eye conditions [14]. Similarly, topical dexamethasone and TAC treatments were found effective in mouse allergic conjunctivitis model to suppress the infiltration of eosinophil and lymphocyte into subconjunctival tissues in mouse [26]. Due to complications associated with corticosteroids, other alternatives are sought for similar therapeutic applications. Latest clinical trials revealed that TAC has equivalent effect as corticosteroids in ocular allergic crisis control and maintenance therapy with very low complications and adverse effects [27], [28], [29].

TAC was found to inhibit the histamine release and its action in addition to the inhibition of CD4-lymphocytes activation. It also inhibited prostaglandin synthesis in mast cells and basophils [15], [30]. Due to its potential to reduce activated T-cells, the TAC efficacy has been investigated for topical ocular use to prevent the corneal graft rejection and hence topical ointment of 0.03% TAC has been evaluated as a second-line treatment in high-risk corneal grafts patients [31].

In the present investigation TAC-loaded PLGA-NPs was formulated by emulsification-diffusion method to determine the ocular bioavailability of TAC after topical administration in rabbit eyes. For comparison 0.03% TAC aqueous suspension (TAC-AqS) was also incorporated in the investigation.

Section snippets

Materials

“Tacrolimus, cyclosporine-A, ploy (d,l-lactide-co-glycolide; lactide: glycolide (50:50)) with molecular weight 30,000–60,000 and Pluronic-F68 (Poloxamer-188) were purchased from Sigma Aldrich Co. (St. Louis MO, USA)”. Polyvinyl alcohol (PVA, Mw 17,200), ethyl acetate and acetone were purchased from “AVONCHEM Ltd. (Wellington House, Waterloo St. West, Macclesfield, Cheshire, UK)”. Chloroform and dichloromethane (DCM) were purchased from MERK (Darmstadt, F.R. Germany) and PANREAC QUIMICA SA,

Data analysis

The TAC concentration was estimated from the drug recovery in the collected samples. By using non-compartmental approach, the pharmacokinetic parameters (t1/2, tmax, Cmax, AUC0-t and AUC0-inf) were computed by a software (PK-Solver, Nanjing, China through MS-Excel-2013) [44]. One-way ANOVA was applied to compare the obtained pharmacokinetic parameters, where p < 0.05 was assumed as statistically significant.

Formulation of TAC-NPs

The TAC-NPs were formulated by emulsification-solvent diffusion method where, PLGA concentration in the internal organic phase was a significant factor in increasing the NPs-size, the size increases with increasing the PLGA concentration. Thus, 1%, w/v, PLGA concentrations was chosen to formulate the small sized NPs based on the reported study [47]. The PVA as stabilizer at 0.5–1.0%, w/v concentrations with or without Pluronic F-68 (0.5–1.0%, w/v) and Tween-80 (0.1%, w/v) were applied to assess

Conclusion

On the basis of the data it can be concluded that the F2 formulation is the best among the lot of five optimized PLGA-NP formulations, in terms of characterization parameters, transcorneal permeation and stability. The Draize’s test did not reveal any irritant or corrosive effects of the F2 and indicates its nontoxic nature when administered topically into the eyes. Comparing the pharmacokinetic data, F2 has shown high TAC ocular bioavailability than that of TAC-AqS. Higher availability of TAC

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

“The authors are thankful to the College of Pharmacy Research Center and the Deanship of Scientific Research at King Saud University for financial support and logistic assistance”.

References (58)

  • Y. Diebold et al.

    Ocular drug delivery by liposome-chitosan nanoparticle complexes (LCS-NP)

    Biomaterials

    (2007)
  • J. Yuan et al.

    Determination of tacrolimus in rabbit aqueous humor by liquid chromatography-electrospray ionization tandem mass spectrometry

    J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.

    (2008)
  • S. Akhter et al.

    Improving the topical ocular pharmacokinetics of an immunosuppressant agent with mucoadhesive nanoemulsions: formulation development in-vitro and in-vivo studies

    Colloids Surf. B: Biointerfaces.

    (2016)
  • Y. Zhang et al.

    PKSolver An add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel

    Comput. Methods Programs Biomed.

    (2010)
  • H.-Y. Kwon et al.

    Preparation of PLGA nanoparticles containing estrogen by emulsification-diffusion method

    Colloids Surf. A: Physicochem. Eng. Asp.

    (2001)
  • H. Murakami et al.

    Influence of the degrees of hydrolyzation and polymerization of poly(vinylalcohol) on the preparation and properties of poly(dl-lactide-co-glycolide) nanoparticle

    Int. J. Pharm.

    (1997)
  • A. Zimmer et al.

    Microspheres and nanoparticles used in ocular delivery systems

    Adv. Drug Del. Rev.

    (1995)
  • R.H. Muller et al.

    Solid lipid nanoparticles (SLN) for controlled drug delivery-a review of the state of the art

    Eur. J. Pharm. Biopharm.

    (2000)
  • H. Murakami et al.

    Preparation of poly(DL-lactide-co-glycolide) nanoparticles by modified spontaneous emulsification solvent diffusion method

    Int. J. Pharm.

    (1999)
  • H. Gupta et al.

    Sparfloxacin-loaded PLGA nanoparticles for sustained ocular drug delivery

    Nanomedicine : NBM

    (2010)
  • E. Knop et al.

    Anatomy and immunology of the ocular surface

    Chem. Immunol. Allergy

    (2007)
  • J.G. Souza et al.

    Topical delivery of ocular therapeutics: carrier systems and physical methods

    J. Pharm. Pharmacol.

    (2014)
  • A. Vasconcelos et al.

    Conjugation of cell-penetrating peptides with poly(lactic-co-glycolic acid)-polyethylene glycol nanoparticles improves ocular drug delivery

    Int. J. Nanomed.

    (2015)
  • A.K. Sah et al.

    PLGA nanoparticles for ocular delivery of loteprednol etabonate: a corneal penetration study

    Artif. Cells Nanomed. Biotechnol.

    (2016)
  • M.G. Qaddoumi et al.

    The characteristics and mechanisms of uptake of PLGA nanoparticles in rabbit conjunctival epithelial cell layers

    Pharm. Res.

    (2004)
  • R.L. McCall et al.

    PLGA nanoparticles formed by single- or double-emulsion with vitamin E-TPGS

    J. Vis. Exp.

    (2013)
  • M.A. Kalam et al.

    Preparation characterization, and evaluation of gatifloxacin loaded solid lipid nanoparticles as colloidal ocular drug delivery system

    J. Drug Target.

    (2010)
  • B.K. Moscovici et al.

    Clinical treatment of dry eye using 0.03% tacrolimus eye drops

    Cornea

    (2012)
  • G.G. Muller et al.

    Topical tacrolimus 0.03% as sole therapy in vernal keratoconjunctivitis: a randomized double-masked study

    Eye Contact Lens.

    (2014)
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