Saffron extract self-assembled nanoparticles to prolong the precorneal residence of crocin

Abstract

Saffron has been widely proposed for the treatment of different ocular pathologies, thanks to the anti-inflammatory properties of crocin and crocetin. The aim of this study was to prepare self-assembling saffron extract nanoparticles (SE-NPs) in the presence of chitosan without the addition of any cross-linker. After lyophilization by adding trehalose, SE-NP resulted spherical, 311.6 ± 7.7 nm in size and having a zeta potential of 25.4 ± 0.9 mV. Furthermore, SE-NP were able to protect both polyphenols and crocin from degradation for at least 4 h in simulated tear fluid. NPs formation was due to hydrogen bonds and hydrophobic interaction between saffron extract and chitosan, as demonstrated by FTIR spectra. Thanks to their positive surface charge, SE-NP were able to increase the residence time of crocin in the precorneal area by 2.7 times compared to free crocin. Since lyophilized SE-NP showed a good storage stability they could represent a manageable formulation for application in the eyes.

Introduction

The main causes of blindness are correlated with neurodegenerative disorders such as glaucoma, retinitis pigmentosa, diabetic retinopathy (DR), etc., of which no efficient therapy is now available. Nevertheless, some natural products, especially thanks to their low toxicity, play an important role in preventing the above neurodegenerative pathologies of the eye. In fact, natural products derived from plants exhibit defense mechanisms against microbic agents, xenobiotics and/or detrimental physical factors. Among agri-food products saffron (dried stigmas of Crocus sativus L.) has widely been proposed for the treatment of different ocular pathologies [1] because its active compounds, such as crocin and crocetin, exhibiting antioxidant properties, could be effective in the treatment of inflammatory diseases [2]. For example, it has been demonstrated that saffron reduces insulin-resistance thus controlling microglia activation in DR [3]. In glaucoma, a saffron extract dose of 30 mg/die produced a decrease in intraocular pressure and, consequently, a retina protection from damage [4]. This protective effect could be related to the anti-inflammatory properties of crocin and crocetin, which scavenge ROS and stimulate the production of endogenous antioxidant enzymes. The ability of crocin to scavenge ROS has indeed been demonstrated with human corneal epithelial cells [5]. Some studies have shown that crocin can protect retina against cell apoptosis thanks to multifactorial-induced oxidative stress [6] and neuroprotective effects on retinal neural cells [7]. In particular, it seems that crocin protects retinal photoreceptors damage caused by light exposure, maintaining their morphology and function and acting as a regulator of programmed cell death [8]. Furthermore, oral administration of crocin has been shown to prevent the damage caused by an increased intraocular pressure in the retinal tissue [9]. Saffron has been shown to counteract the harmful effects on retina by the same neuroprotection mechanism as cannabinoids [10].

However, high doses are required for orally administered saffron to exert therapeutic effects, while it is well-documented that adverse effects (e.g., nausea, diarrhea, vomit, etc.) occur at as low doses as 5 g/die. Actually, a 20 g/die dose could be lethal [11].

The main constituent of saffron stigmas, crocin, has poor storage stability and is prone to degradation, as it contains multiple double bonds [12]. Numerous studies have been carried out to improve the stability of crocin by incapsulating it in nanosystems [[13], [14], [15]]. For example, chitosan/alginate nanoparticles entrapping crocin have shown to enhance the stability of crocin itself into gastrointestinal tract exhibiting antioxidant and anticancer properties [13,16]. Crocin-solid lipid nanoparticles showed a more prolonged antioxidant activity compared to free crocin [17].

The potential of nanosystems in ocular delivery has extensively been reported in the literature [18]. One of the polymers of choice to prepare colloidal drug carriers is chitosan (Ch) and its derivatives, indeed, chitosan-based nanosystems interact with corneal or conjunctival epithelium cells [19]. Once internalized by epithelial cells, these nanosystems might make the conjunctival and corneal epithelia reservoirs for drug delivery to the exterior or interior of the eye [20]. In addition, Ch, in virtue of its proved safety, has been approved by the Food and Drug Administration (FDA) for the preparation of functional foods.

Recently, nanoparticles (NPs) based on Ch and hyaluronic acid were developed for the potential release of crocin and actinoquinol in the eye demonstrating in vitro a good ability to promote the absorption of the active ingredients [21]. For this reason, we decided to optimize the conditions for the preparation of self-assembling saffron extract nanoparticles (SE-NP) in the presence of chitosan without the addition of any cross-linker. In fact, the objective of this study has been to develop an ocular formulation based on self-assembling and mucoadhesive nanoparticles to prolong the residence time of the crocin contained in the saffron extract in the precorneal area. In the present study, the nature of the interaction between SE and Ch to form self-assembling nanoparticles, as well as the ability of SE-NP to protect crocin from degradation and to improve the precorneal residence time in the rabbit eyes have been investigated.