Corneal targeted Sertaconazole nitrate loaded cubosomes: Preparation, statistical optimization, in vitro characterization, ex vivo permeation and in vivo studies

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Abstract

Sertaconazole nitrate (STZ) is a poorly soluble antifungal drug commonly used for treating fungal skin infections. Introducing it as a new treatment option for the management of fungal keratitis, requires the development of a delivery system capable of targeting the infected cornea with an adequate STZ concentration. Hence, Sertaconazole nitrate loaded cubosomes (STZ-CUBs) were prepared, characterized and optimized based on a 33 central composite face-centred design. Optimized formulation (CUB-opt) showed maximum desirability (0.905), with solubilization efficiency (SE%) of 94.50 ± 0.51%, particle size (PS) of 216.55 ± 2.33 nm, polydispersity index (PDI) of 0.229 ± 0.11 and zeta potential (ZP) of 34.00 ± 6.93 mV. Under the transmission electron microscope, it showed discrete cubic shaped structures. Moreover, it exhibited a promising mucoadhesive behavior, terminal sterilization stability, and storage stability. Ex vivo corneal permeation study revealed its ability to enhance the steady state flux (Jss) and the permeability coefficient (KP) of STZ, compared to STZ-suspension. Finally, CUB-opt formulation was found to be safe on the corneal tissues in the in vivo corneal tolerance study, and demonstrated a superior corneal penetration power in the in vivo corneal uptake study.

Introduction

Fungal keratitis is a serious corneal infection caused by fungal species capable of invading the surface of the eye such as Fusarium, Aspergillus or Candida (Thomas and Kaliamurthy, 2013). It should be effectively treated without delay or else it may cause severe corneal ulceration, which is the second leading cause of loss of vision after cataract (Singh et al., 2017). Treating fungal infections is challenging since antifungal agents most often interfere with fungal cell membrane biogenesis, a process that is commonly shared between eukaryotic cells (Mazu et al., 2016). This problem can be overcome by the use of antifungals that target cellular components and processes exclusive to fungi, and the use of targeted delivery systems that deliver antifungals with proper concentration to the exact site of infection (Mor et al., 2015, Walsh et al., 2000).

Sertaconazole nitrate (STZ) is an imidazole antifungal agent, whose fungistatic activity is mediated by the inhibition of 1,4-α demethylase enzyme, preventing the formation of ergosterol necessary for fungal cell membrane formation, boosting the cellular permeability to the extent of leaking the cellular components (Croxtall and Plosker, 2009). STZ is most commonly used for the treatment of skin, hair and nail fungal infections (Carrillo-Munoz et al., 2013). Yet, its broad spectrum antifungal activity, its anti-inflammatory activity, and good ocular tolerance allows it to be used as an effective treatment for fungal keratitis (Romero et al., 1996, Sur et al., 2008). However, its poor aqueous solubility stands as an obstacle against its formulation as conventional eye drops (Perdomo-López et al., 2002). Therefore, the challenge is to formulate STZ in a suitable delivery system that could target the infection site in the eye with a suitable concentration.

Administration of antifungal drugs topically is the most common choice when treating fungal keratitis (Al-Badriyeh et al., 2009), due to simplicity of instillation, non-invasiveness, rapid onset of action, low cost, low systemic side effects and high patient acceptability (Davies, 2000). Topically administered drugs penetrate the ocular tissues mainly through the transcorneal route (Addo, 2016). Achieving the desired bioavailability of antifungals through this route is challenging due to the barrier nature of the cornea as well as the poor physicochemical characteristics of the applied drugs (Durairaj, 2016). To address these challenges, several non-conventional drug delivery systems have been created, such as lipid nanoparticles (Sánchez-López et al., 2017), microemulsions (Kalam et al., 2016), micelles (Di Prima et al., 2017) and in situ gels (Fathalla et al., 2017).

In the last few years, monoolein (MO)-based nanosystems such as cubosomes have gained growing interest as ocular drug delivery systems (Huang et al., 2017, Verma and Ahuja, 2016). This can be attributed to their high drug loading for drugs of diverse hydrophilicities, biocompatibility, enhanced drug stability, controlled drug release, bio-adhesion, enhanced transcorneal permeability and prolonged corneal retention (Ali et al., 2016, Chen et al., 2014, Gan et al., 2010). Cubosomes have a unique structure of two continuous but nonintersecting water channels separated by a contorted lipid bilayer, created from the fragmentation of cubic liquid crystalline bulk phases in excess aqueous media (Huang et al., 2017, Spicer et al., 2003). However, cubosomal dispersions are not colloidally stable without the use of stabilizers (Gustafsson et al., 1999). The efficacy of these stabilizers depends on their ability to provide stabilisation without altering the internal structure of cubosomes (Chong et al., 2011). The most commonly used stabilizers are non-ionic block copolymers as Pluronic, Tween 80, Cremophor…etc (Chong et al., 2015, Elnaggar et al., 2015).

Terpenes are a series of naturally occurring compounds composed of multiple isoprene (C5H8) units. In recent years, they have gained much attention in the pharmaceutical field as penetration enhancers (Dragicevic et al., 2015, Williams and Barry, 2004). Limonene is a hydrocarbon monoterpene, employed as a penetration enhancer in several skin permeation studies and demonstrated superiority over other counterparts (Charoenputtakun et al., 2014, Inoue et al., 2008). Its remarkable permeation enhancement properties as well as its anti-inflammatory effect (Chaudhary et al., 2012, Hirota et al., 2010), qualifies it to be used as a penetration enhancer in ocular drug delivery systems.

Earlier literature lacks adequate data about the inclusion of STZ in ocular drug delivery systems despite its potential usefulness as a treatment for fungal keratitis. Also, up to date, the use of limonene (hydrophobic terpene) as a safe penetration enhancer in the ocular route has not been investigated yet. Thus, our aim in this study was to develop cubosomes enriched with limonene as nanocarrier systems that can overcome the poor solubility of STZ and would provide acceptable ocular tolerance, adequate stability, and enhanced transcorneal permeation. To this end, initially, a screening was performed to select the most suitable aqueous and lipid phase stabilizers. Then, a 33 central composite face-centred design was used to prepare STZ-loaded cubosomes (STZ-CUBs), to study the effect of different formulation variables on the desired dependent variables, and to optimize the formulations according to the desirability value. Finally, the elucidated optimized formulation was subjected to morphological examination, several in vitro studies, ex vivo permeation study, a stability study and in vivo studies (ocular tolerance and ocular uptake).

Section snippets

Materials

Sertaconazole nitrate (STZ) was supplied as a kind gift by October Pharma, 6th of October City, Egypt. DL-α-Monoolein (MO), Pluronic® F127, Brij® 58, Pluronic F108, Tween® 80, Poly (vinyl alcohol) (PVA; Mwt. 13,000–23,000, 87–89% hydrolyzed), Mucin from porcine stomach and Rhodamine B (RhB) were purchased from Sigma-Aldrich® Inc.AL, USA. (R)-(+)-Limonene, 97% was purchased from Alfa Aesar, Johnson Matthey Chemicals, USA. Ethanol (95%), sodium bicarbonate, sodium chloride and calcium chloride

Selection of stabilizers

All used stabilizers produced milky white cubosomal dispersions with no phase separation or visible aggregates, so they were evaluated for their solubilization efficiency (SE%). ANOVA-factorial demonstrated that changing the type of both the lipid phase stabilizer and aqueous phase stabilizer had significant effects on SE% (p = 0.0008 and p < 0.0001, respectively). Changing the type of lipid phase stabilizer from Pluronic® F127 to Pluronic® F108 significantly decreased SE%. This might be

Conclusion

In this study, Pluronic® F127 and Brij® 58 were selected as the lipid phase stabilizer and the aqueous phase stabilizer, respectively. Sertaconazole nitrate loaded cubosomes (STZ-CUBs) were successfully developed according to a 33 CCFD and optimized via the desirability function. The optimized formulation (CUB-opt) was found to be nanometric in range and cubical in shape. The in vitro results demonstrated its ability to withstand sterilization with gamma irradiation, its promising mucoadhesive

Disclosure statement

The authors report no conflicts of interest.

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