New formulation of vasoactive intestinal peptide using liposomes in hyaluronic acid gel for uveitis

doi:10.1016/j.jconrel.2009.05.033

Journal of Controlled Release

Volume 139, Issue 1, 1 October 2009, Pages 22-30

https://doi.org/10.1016/j.jconrel.2009.05.033 Get rights and content

Abstract

We evaluated the benefits of a novel formulation of vasoactive intestinal peptide (VIP) based on the incorporation of VIP-loaded rhodamine-conjugated liposomes (VIP-Rh-Lip) within hyaluronic acid (HA) gel (Gel-VIP-Rh-Lip) for the treatment of endotoxin-induced uveitis (EIU) in comparison with VIP-Rh-Lip alone. In vitro release study and rheological analysis showed that interactions between HA chains and liposomes resulted in increased viscosity and reinforced elasticity of the gel. In vivo a single intravitreal injection of Gel-VIP-Rh-Lip was performed in rats 7 days prior to uveitis induction by subcutaneous lipopolysaccharide injection. The maximal ocular inflammation occurs within 16–24 h in controls (VIP-Rh-Lip, unloaded-Rh-Lip). Whereas intraocular injection of VIP-Rh-Lip had no effect on EIU severity compared with controls, Gel-VIP-Rh-Lip reduced significantly the clinical score and number of inflammatory cells infiltrating the eye. The fate of liposomes, VIP and HA in the eyes, regional and inguinal lymph nodes and spleen was analyzed by immunostaining and fluorescence microscopy. Retention of liposomes by HA gel was observed in vitro and in vivo. Inflammation severity seemed to impact on system stability resulting in the delayed release of VIP. Thus, HA gel containing VIP-Rh-Lip is an efficient strategy to obtain a sustained delivery of VIP in ocular and lymph node tissues.

Graphical abstract

Introduction

Human uveitis is an intraocular inflammatory disease, associated or not with systemic inflammation, and characterized by inflammatory attack of the uvea and the neuro-retina, resulting in severe loss of vision and morbidity. In clinical practice, the use of systemic corticoids and/or immunosuppressive agents is frequently complicated by systemic side effects [1], [2]. In order to reduce the use of systemic corticotherapy, new drug delivery systems have been developed and a non-biodegradable implant, releasing fluocinolone acetonide for several years is now available (Retisert® Bauch et Lomb, USA) [3], [4]. However, whilst systemic side effects of corticosteroids are avoided, the ocular complications, such as cataract and intraocular pression raise are frequent. This device requires a surgical procedure for intraocular implantation and might be retrieved when empty. Finally, long-term exposure to glucocorticoids may have toxic effects on retinal cells through newly characterized cell death pathways [5]. Therefore, there still is a need to develop more specific compounds to modulate the ocular immune response, delivered by biodegradable and injectable well-tolerated materials, avoiding surgical procedures. Among the new strategies investigated to treat experimental ocular inflammation, we demonstrated that a single intravitreal injection of vasoactive intestinal peptide (VIP) as a new immunosuppressive agent was efficient at reducing clinical and pathological ocular inflammation signs, when injected simultaneously to induction of endotoxin-induced uveitis (EIU) and experimental autoimmune uveoretinitis in Lewis rats. In experimental autoimmune uveoretinitis, the treatment was also effective when started 6 days after immunization, i.e. not far from the onset of the intraocular immune response. Nevertheless, the therapeutic effect was only obtained when VIP was encapsulated in liposomes [6], [7]. Moreover, liposomes are easily injectable and less invasive than surgical implantation. Indeed, this formulation enhanced the immunosuppressive effect of VIP by preserving its integrity and controlling its delivery to all tissues affected by or involved in ocular inflammation [6], [7]. After a unique intravitreal injection, liposomes leak rapidly from ocular tissues through lymphatic and venous circulation, which leads to the presence of liposomes and intact VIP in regional lymph nodes, and in a lesser extent, in inguinal lymph nodes and spleen [6], [8]. However, the leakage of VIP-loaded liposomes from the eye could reduce the long-term efficacy of VIP in ocular tissues. To maintain efficient VIP concentration within the eye, repeated intraocular injections may be necessary, possibly leading to adverse effects and complications [9].

The aim of the present study was to develop an original liposome-based formulation allowing sustained delivery of VIP and to evaluate its potential for the treatment of uveitis. The incorporation of liposomes in hydrogels is an interesting strategy to delay and control the release of drugs for local delivery [10], [11], [12]. In the present study, VIP-loaded liposomes were dispersed within a gel, in order to increase the residence time of liposomes in the eye, thus allowing a prolonged presence of intact VIP in ocular tissues, and possibly reinforce local versus systemic effect of the treatment. The simple hydration of the polymer with VIP-Rh-Lip also presents the advantage of avoiding the use of organic solvent and mechanic stress that could be deleterious for liposomes or VIP. Moreover, polymer concentration in the formulation could be modulated and the concentration of VIP that was previously and successfully used for incorporation of VIP within liposomes [7] can be maintained. Hyaluronic acid (HA) was selected in this study. This negatively charged polysaccharide composed of d-glucuronic acid and N-acetyl-d-glucosamine, is the main component of the vitreous (65 to 400 µg/mL) [13], [14] and is currently used for visco-supplementation during ocular surgery. It was also shown that when using hyaluronic acid for posterior segment surgery, no intraocular pressure raise was observed [15]. Moreover, the intraocular injection of gentamicin formulated in HA gel resulted in increased half-life in vitrectomized eyes, leading to a therapeutic effect on endophthalmitis [16]. Furthermore, high molecular weight HA could have by itself anti-inflammatory properties that could be synergistic with those of VIP [17].

In this study, the effect of HA gel containing VIP-loaded liposomes was evaluated on the severity of LPS-induced ocular inflammation in rats (EIU model). First, in vitro studies were performed to characterize HA gel containing liposomes. In order to demonstrate that incorporation of VIP-loaded liposomes in HA gel allowed a sustained release of VIP-Rh-Lip, intravitreal injection of the formulations was performed 7 days prior to uveitis induction, the maximal inflammation occurring 16–24 h after LPS subcutaneous injection. The fate of liposomes, VIP and hyaluronic acid in the eyes and regional draining lymph nodes was analyzed.

Section snippets

Materials

VIP (3326 g/mol) was provided by Bachem AG (Bubendorf, Swiss). Egg phosphatidylcholine (PC) was obtained from Lipoid GmbH (Luwigshafen, Germany). Egg yolk phosphatidylglycerol (PG), cholesterol (Chol), trehalose, fluoresceinamine, cyclohexil isocyanide, dimethyl sulfoxide (DMSO), calcein (622 g/mol), 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid (HEPES) and sodium dodecyl sulfate (SDS) were purchased from Sigma-Aldrich Co (St Louis, USA).

Physicochemical characterization of liposomes

VIP-Rh-Lip and U-Rh-Lip were rather polydisperse (PI = 0.3), between 300 and 600 nm for VIP-Rh-Lip and between 250 and 400 nm for Rh-Lip. Calcein-Lip and U-Lip had a smaller polydispersity index (PI ≤ 0.2), with hydrodynamic mean diameters of 237 ± 60 nm and 175 ± 73 nm respectively. Negative zeta potential values between − 28 and − 38 mV were found for all formulations. VIP and calcein encapsulation efficiencies were 3.3 and 20 mmol/mol lipids respectively.

Release study of Calcein-Lip from HA gel

After gel injection in HEPES/NaCl buffer, gel

Discussion

The aim of this work was to evaluate whether a novel formulation of VIP, based on the incorporation of VIP-loaded liposomes within HA gel, allows the retention of liposomes-VIP in the vitreous leading to a long lasting delivery of VIP from this formulation. This new formulation would thus present benefits for the treatment of endotoxin-induced uveitis (EIU) in comparison with VIP-loaded liposomes alone. During cataract surgery, raise of intraocular pressure after hyaluronic acid injection in

Conclusion

In the present study, we show that HA gel formulation of liposomes loaded with VIP offers a one-week-delayed efficacy on ocular inflammation. High viscosity and interactions between liposomes and HA gel allow for a sustained delivery of VIP in ocular tissues and in regional lymph nodes. HA gel formulation of liposomes appears as a good candidate for the delivery of therapeutic molecules that would be useful in the treatment of chronic ocular pathologies such as autoimmune uveitis.

Acknowledgements

Helpful advice and technical assistance were kindly provided by Christophe Klein, in charge of confocal microscopy from the Centre de Recherche des Cordeliers (Université Pierre et Marie Curie Paris 6, UMR S 872). The authors would like to thank Karine Andrieux (UMR CNRS 8612, Faculté de Pharmacie, Châtenay-Malabry) for technical assistance concerning calcein assay.

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New formulation of vasoactive intestinal peptide using liposomes in hyaluronic acid gel for uveitis

Abstract

Graphical abstract

Introduction

Section snippets

Materials

Physicochemical characterization of liposomes

Release study of Calcein-Lip from HA gel

Discussion

Conclusion

Acknowledgements

Ophthalmology

Colloids Surf., B Biointerfaces

Trends Immunol.

J. Biol. Chem.

Eur. J. Pharm. Biopharm.

J. Neuroimmunol.

Trends Immunol.

Trends Immunol.

Int. J. Pharm.

J. Colloid Interface Sci.

Colloids Surf., A

J. Struct. Biol.

Exp. Eye Res.

Immunotherapy of uveitis: is gene therapy in our future?

Diagnosis and Treatment of Uveitis

Retisert: is the new advance in treatment of uveitis a good one?

Ann. Pharmacother.

Glucocorticoids induce retinal toxicity through mechanisms mainly associated with paraptosis

Mol. Vis.

Protective effect of intravitreal injection of vasoactive intestinal peptide (VIP)-loaded liposomes on experimental autoimmune uveoretinitis

J. Ocular Pharmacol. Ther.

Downregulation of endotoxin-induced uveitis by intravitreal injection of vasoactive intestinal peptide encapsulated in liposomes

Invest. Ophthalmol. Vis. Sci.

Drainage of fluorescent liposomes from the vitreous to cervical lymph nodes via conjunctival lymphatics

Ophthalmic Res.