Biopharmaceutical evaluation of epigallocatechin gallate-loaded cationic lipid nanoparticles (EGCG-LNs): In vivo, in vitro and ex vivo studies

https://doi.org/10.1016/j.ijpharm.2016.02.039Get rights and content

Abstract

Cationic lipid nanoparticles (LNs) have been tested for sustained release and site-specific targeting of epigallocatechin gallate (EGCG), a potential polyphenol with improved pharmacological profile for the treatment of ocular pathologies, such as age-related macular edema, diabetic retinopathy, and inflammatory disorders. Cationic EGCG-LNs were produced by double-emulsion technique; the in vitro release study was performed in a dialysis bag, followed by the drug assay using a previously validated RP-HPLC method. In vitro HET-CAM study was carried out using chicken embryos to determine the potential risk of irritation of the developed formulations. Ex vivo permeation profile was assessed using rabbit cornea and sclera isolated and mounted in Franz diffusion cells. The results show that the use of cationic LNs provides a prolonged EGCG release, following a Boltzmann sigmoidal profile. In addition, EGCG was successfully quantified in both tested ocular tissues, demonstrating the ability of these formulations to reach both anterior and posterior segment of the eye. The pharmacokinetic study of the corneal permeation showed a first order kinetics for both cationic formulations, while EGCG-cetyltrimethylammonium bromide (CTAB) LNs followed a Boltzmann sigmoidal profile and EGCG-dimethyldioctadecylammonium bromide (DDAB) LNs a first order profile. Our studies also proved the safety and non-irritant nature of the developed LNs. Thus, loading EGCG in cationic LNs is recognised as a promising strategy for the treatment of ocular diseases related to anti-oxidant and anti-inflammatory pathways.

Graphical abstract

Transcorneal permeation profile of EGCG-CTAB LNs (■) and EGCG-DDAB LNs (♦), and respective stereomicrographs of the CAM after 5 min of exposure to lipid nanoparticles.

  1. Download : Download high-res image (99KB)
  2. Download : Download full-size image

Introduction

The anatomical and physiological barriers that ocular globe offers against the entrance of drugs are a challenge that could be overcome by the use of nanoparticles (Abrego et al., 2015, Fangueiro et al., 2015, Fangueiro et al., 2016). The main barriers encountered in ocular drug delivery are the physical barriers (cornea, sclera and retina), the blood aqueous and blood-retinal barriers, and the physiological barriers (blood flow, lymphatic clearance, enzymatic degradation, protein binding and the tear dilution and renovation), compromising the delivery of drugs specially targeted to the posterior segment of the eye (Fangueiro et al., 2016).

Topical application of drugs in the ocular mucosa is usually used to reach the anterior and/or posterior segments of the eye (Araujo et al., 2011). The anterior segment of the eye is anatomically composed of the cornea, conjunctiva, sclera and anterior uvea. Usually, the most commonly applied formulations to reach these structures are the eye drops, which are rapidly drained from the ocular surface reducing drastically drug residence time (Urtti, 2006). The posterior segment of the eye is composed of retina, vitreous and choroid (Thrimawithana et al., 2011). Because of their anatomic location, the pathologies affecting these structures are difficult to treat. To reach them, invasive and painful approaches (e.g. intravitreal injection) are usually required for the delivery of high drug doses. However, intravitreal injection can cause complications, such as endophthalmitis and, because of the administered high doses, systemic absorption may be compromised (Kim et al., 2014). These pathologies include age-related macular degeneration (AMD), retinitis pigmentosa, diabetic retinopathies (DR), and neural consequences caused by glaucoma (Kim et al., 2014, Urtti, 2006).

Designing non-invasive and sustained delivery nanoparticles for targeting drugs to the anterior and posterior segment of the eye is an ultimate objective of our work. Cationic lipid nanoparticles (LNs) for ocular delivery have been reported to be safe, and long-term stable (Fangueiro et al., 2014a, Fangueiro et al., 2014b). The use of physiological lipids is being documented as a safe, biocompatible and biodegradable approach (Souto et al., 2013). However, the main concern is the toxicity related to the surfactants and, in this particularly case, to the use of cationic molecules. We have recently produced biocompatible and biodegradable cationic epigallocatechin gallate LNs (EGCG-LNs) (Fangueiro et al., 2014a, Fangueiro et al., 2014b), composed of natural and physiological lipids, cationic lipid, surfactants, EGCG and water. LNs have been widely exploited for targeted drug delivery because of their nanometer size, long-term physicochemical stability, biocompatibility and biodegradability, controlled release of drugs, low cytotoxicity and easily scale-up production (Mishra et al., 2010, Müller et al., 2000, Sinha et al., 2011, Souto and Muller, 2010, Souto and Müller, 2007, Souto et al., 2010, Vazzana et al., 2015). It is therefore anticipated that LNs could overcome some of the disadvantages of ocular drug delivery and could provide a modified release profile (controlled/prolonged), keeping the drug concentration within the therapeutic levels, over an extended time (Araujo et al., 2009).

The topical administration of drugs provides a short period of residence time in the mucosa (usually 1–2 min), and these are efficiently removed via the lacrimal fluid renovation (Araujo et al., 2009). Cationic LNs seem to offer high capacity to increase the bioavailability of topical drugs when administered onto the ocular mucosa since they promote electrostatic interactions between the surface of the cationic particles and the anionic ocular mucosa, with a considerable improvement of the drug residence time (Fangueiro et al., 2014a).

The polyphenol EGCG with anti-oxidant properties has been successfully formulated in cationic LNs (Fangueiro et al., 2014b). Currently, there are no studies involving the topical use of this drug to treat ocular diseases such as DR, diabetic macular edema (DME), glaucoma or AMD. Some of the biological mechanisms involved in the development of these ocular pathologies are related to the increased oxidative stress due to the inability of the organism to suppress the production of reactive oxygen species (ROS) (Kowluru and Chan, 2007, Madsen-Bouterse and Kowluru, 2008, Wakamatsu et al., 2008). It is well-known that polyphenols have potential human health benefits (Heim et al., 2002), and recently EGCG is being reported as one of the most potent polyphenols (Du et al., 2012, Yamauchi et al., 2009). Our group stabilized EGCG in biological medium for further characterization of the physicochemical stability and release/permeation profile under physiological conditions (Fangueiro et al., 2014c).

Our efforts have been driven towards the development of cationic LN formulations that should maximize the EGCG ocular drug absorption via prolonged drug residence time in the cornea and conjunctival sac, as well as to enhance the transcorneal and transscleral drug penetration. In the present paper, the ex vivo transcorneal and transscleral permeation profiles of the EGCG-LNs have been evaluated, in parallel with the in vitro release profile of EGCG from the LNs. Pharmacokinetic parameters for each tissue were determined to establish the mechanism for transcorneal and transscleral permeation of the EGCG. In order to assess the local LNs toxicity and irritancy, an in vitro test (HET-CAM test) and an in vivo test (Draize test) have been carried out.

Section snippets

Materials

Epigallocatechin gallate (EGCG, 98% purity, molecular weight 458.7 g/mol and pKa 7.59–7.75) was purchased from CapotChem (Hangzhou, China). Softisan®100 (S100) was a free sample from Sasol Germany GmbH (Witten, Germany), Lipoid®S75, 75% soybean phosphatidylcholine was purchased from Lipoid GmbH (Ludwigshafen, Germany), Lutrol®F68 or Poloxamer 188 (P188) was a free sample from BASF (Ludwigshafen, Germany). Transcutol®P was a gift from Gattefossé (Barcelona, Spain). Acetic acid (glacial, AR

Results and discussion

Cationic LNs were previously optimised by a factorial design study (Fangueiro et al., 2014a) and physicochemically characterised for ocular delivery (Fangueiro et al., 2014b). The DLS studies showed cationic LNs with a mean particle size for both EGCG-LNs below 150 nm and a PI below 0.25 (Table 2).

However, further studies employing other techniques were carried out since ocular delivery requires a narrow control in the particle size, to avoid possible irritations and lesions to the eye (Araujo

Conclusion

The cationic EGCG-LNs for ocular delivery revealed promising results and may be an innovative approach for the treatment of several eye disorders, such as AMD, DR and glaucoma. To complement our previous studies, reporting the good physicochemical parameters, the present work provides additional information related to the release of EGCG. The LNs provide a prolonged release of EGCG, following the Boltzmann sigmoidal release profile. The ex vivo permeation of EGCG was achieved in the cornea and

Acknowledgements

Ms. Joana Fangueiro wishes to acknowledge Fundação para a Ciência e Tecnologia do Ministério da Ciência e Tecnologia (FCT, Portugal) and Programa Operacional Potencial Humano (POPH/QREN) under the reference SFRH/BD/80335/2011. FCT and European Funds (FEDER and COMPETE) are also acknowledged under the research project FCOMP-01-0124-FEDER-022696 (UID/AGR/04033/2013). The Subprograma de Proyectos de Investigación Fundamental no Orientada del Ministerio de Ciencia e Innovación is also acknowledged

References (48)

  • J.F. Fangueiro et al.

    Current nanotechnology approaches for the treatment and management of diabetic retinopathy

    Eur. J. Pharm. Biopharm.

    (2015)
  • L. Gilleron et al.

    Evaluation of a modified HET-CAM assay as a screening test for eye irritancy

    Toxicol. In Vitro

    (1996)
  • K.E. Heim et al.

    Flavonoid antioxidants: chemistry, metabolism and structure-activity relationships

    J. Nutr. Biochem.

    (2002)
  • Y.C. Kim et al.

    Ocular delivery of macromolecules

    J. Control. Release

    (2014)
  • J. Li et al.

    A potential carrier based on liquid crystal nanoparticles for ophthalmic delivery of pilocarpine nitrate

    Int. J. Pharm.

    (2013)
  • R.H. Müller et al.

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

    Eur. J. Pharm. Biopharm.

    (2000)
  • B. Mishra et al.

    Colloidal nanocarriers: a review on formulation technology, types and applications toward targeted drug delivery

    Nanomedicine

    (2010)
  • M.R. Prausnitz et al.

    Permeability of cornea, sclera, and conjunctiva: a literature analysis for drug delivery to the eye

    J. Pharm. Sci.

    (1998)
  • E.B. Souto et al.

    Chapter 6—Solid lipid nanoparticle formulations pharmacokinetic and biopharmaceutical aspects in drug delivery

    Methods Enzymol.

    (2009)
  • N. Srikantha et al.

    Influence of molecular shape, conformability, net surface charge, and tissue interaction on transscleral macromolecular diffusion

    Exp. Eye Res.

    (2012)
  • T.R. Thrimawithana et al.

    Drug delivery to the posterior segment of the eye

    Drug Discov. Today

    (2011)
  • A. Urtti

    Challenges and obstacles of ocular pharmacokinetics and drug delivery

    Adv. Drug Deliv. Rev.

    (2006)
  • A. Vargas et al.

    The chick embryo and its chorioallantoic membrane (CAM) for the in vivo evaluation of drug delivery systems

    Adv. Drug Deliv. Rev.

    (2007)
  • M. Vazzana et al.

    Tramadol hydrochloride: pharmacokinetics, pharmacodynamics, adverse side effects, co-administration of drugs and new drug delivery systems

    Biomed. Pharm.

    (2015)
  • Cited by (101)

    View all citing articles on Scopus
    View full text