Life Sciences 216 (2019) 39–48 Contents lists available at ScienceDirect Life Sciences journal homepage: www.elsevier.com/locate/lifescie Protective efficacy of crocetin and its nanoformulation against cyclosporine A-mediated toxicity in human embryonic kidney cells T ⁎ Jyotsnarani Pradhana,b, Chandana Mohantya,1, Sanjeeb K. Sahooa, a Institute of Life Sciences, Bhubaneswar, Odisha, India b P.G. Department of Biotechnology, Utkal University, Bhubaneswar, Odisha, India ARTICLE INFO ABSTRACT Keywords: Aim: This study is aimed to formulate crocetin-loaded lipid Nanoparticles (NPs) and to evaluate its antioxidant Crocetin properties in a cyclosporine A-mediated toxicity in Human Embryonic Kidney (HEK-293) cells in vitro. Nanoparticle Main methods: Crocetin-loaded NPs were prepared followed by physicochemical characterization. In vitro pro- Cyclosporine tective efficacy of crocetin and crocetin loaded NPs was investigated in cyclosporine A-mediated toxicity in HEK- Cytotoxicity 293 cells by assessing free radical scavenging, DNA Nicking, cytotoxicity, intracellular Reactive oxygen species ROS (ROS) inhibition, Mitochondrial membrane potential (MMPs) loss and evaluating the activity and expression of Antioxidant activity antioxidant enzymes and non-enzyme level. Further, we have studied the mechanism of protective activity of crocetin either native or in NPs by studying the expression of phase II detoxifying proteins (HO-1) via Nrf2 mediated regulation. Key findings: Our results showed that pretreatment with crocetin and crocetin-loaded NPs attenuated the cy- closporine A-mediated toxicity, ROS production and exhibited enhance free radical scavenging ability and cy- toprotective activity. Further, the treatment prevented MMPs loss by directly scavenging the ROS and restored the antioxidant enzyme network with normalization of heme oxygenase-1 (HO-1) expression by inhibiting nu- clear translocation of Nrf2. Significance: Pretreatment of crocetin and crocetin-loaded NPs provided pronounce protective effect against cyclosporine A-mediated toxicity in HEK-293 cells by nullifying the ROS formation and restored antioxidant network through inhibition of Nrf2 translocation and followed by expression of HO-1. Such an approach may be anticipated to be beneficial for antioxidant therapy. 1. Introduction food derived antioxidants owing to their therapeutic values, no side effects and of their economic availability [9]. Cyclosporine A (CsA) is an effective immunosupressor commonly Crocetin (8,8′-diapocarotene-8,8′-dioic acid) is a bioactive low used in organ transplantation and in the treatment of autoimmune molecular weight natural carotenoid compound which originates from disorders during the past 30 years [1]. Cyclosporine exerts its im- saffron stigmas (Crocus sativus L.) and gardenia fruit (Gardenia jasmi- munosuppressive action by inhibiting the enzyme calcineurin phos- noides Ellis) mostly used for the treatment of various diseases in tradi- phatase and thereby inhibits the expression of cytokines such as IL2 and tional and modern medicine [10]. Crocetin is reported to have a wide IFN ϒ and subsequently T cell proliferation [2–4]. However, extensive range of pharmacological properties, including anticancer [11], anti- use of CsA leads to severe side effects such as nephrotoxicity, hepato- hyperlipidemia [12], anti-atherosclerosis [13] activities, however, its toxicity and cardiotoxicity [5,6]. Several lines of evidences indicate mechanism of action is not clear. In addition to this, study have shown towards the role of ROS and decreased antioxidant enzyme activity as on numerous in vitro and in vivo animal models yet also confirmed that one of the major culprits of nephrotoxicity induced by CsA [7,8]. To crocetin can also ameliorate the effect of oxidative stress due to its this end, the antioxidant therapy is an effective strategy, which pos- potential antioxidant activity [14]. It can inhibit ROS production and sesses the ability to protect different vital organs, from damage caused inflammatory cascades towards amelioration of cardiac injury caused by the ROS. In this context current research is now focusing on natural by haemorrhage/resuscitation [15]. Ahmad et al. [16] reported that the ⁎ Corresponding author at: Laboratory of Nanomedicine, Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar, Orissa, India. E-mail address: [email protected] (S.K. Sahoo). 1 Present Address: School of Applied Sciences, KIIT Deemed to be University. https://doi.org/10.1016/j.lfs.2018.11.027 Received 24 April 2018; Received in revised form 23 October 2018; Accepted 12 November 2018 Available online 13 November 2018 0024-3205/ © 2018 Elsevier Inc. All rights reserved. J. Pradhan et al. Life Sciences 216 (2019) 39–48 protective effect of crocetin against hemi-parkinsonian in a rat model methods (−47 °C and < 10 μm mercury pressure, Freezone 12, Lab- might be due to reduction in auto-oxidation of dopamine by augmen- conco, Kansas City, MO) to get lyophilized powder for further use. tation of antioxidant enzymes like Glutathione Peroxidase (GPx), Su- peroxide dismutase (SOD), Glutathione S-transferase [16]. However, 2.3. Physical characterization of crocetin-loaded NPs despite its several therapeutic applications its pharmacological activ- ities in clinical settings were hindered due to the oxidative degradation 2.3.1. Particle size analysis and zeta potential measurement of the drug by different external factors which, promotes isomerization Briefly, ~1 mg/ml of crocetin-loaded NPs solution was prepared in of trans-form to cis-form rendering the drug inactive [17,18]. double distilled water (DW). A total of 100 μl of the above solution In this regard, novel drug delivery system (NDDS) can significantly diluted in 1 ml DW, was sonicated for 30 s in an ice bath and taken for improve the ability of drugs in terms of efficacy, solubility, half-life in particle size and zeta potential measurement by in a zetasizer (Nano ZS, blood and bioavailability [19]. One of the important NDDS is nano- ZEN3600, Malvern Instrument, UK). All measurements were performed particle-based drug delivery system, which is emerging as a highly in triplicates. promising technology in escalating drug delivery by improving the drug pharmacokinetics and enabling to overcome the drawbacks of native 2.3.2. Transmission electron microscope (TEM) drugs. By using nanoparticulate drug delivery system different groups The size of the crocetin-loaded NPs was evaluated by TEM (Phillips/ have shown that solubility, bioavailability and therapeutic efficacy of FEI Inc., Briarcliff Manor, NY). For the analysis, a drop of diluted so- various water insoluble herbal compounds such as ellagic acids [20], lution of crocetin-loaded NPs (~1 mg/ml in water) was placed in a quercetin [21], naringenin [22], kaempferol [23] and Coenzyme Q10 carbon-coated copper TEM grid (150 mesh, Ted Pella Inc., CA, USA) [24] can be improved. In this regards, lipid based nanoparticulate and allowed to air dry and the images were visualized at 120 kV under systems have drawn significant attention due to their small and narrow transmission electron microscope. size range (10–200 nm), low intrinsic cytotoxicity and biodegradability. Recently much attention was given to glycerol monooleate (GMO), a 2.3.3. Assessing the entrapment efficiency of crocetin in crocetin-loaded synthetic non-toxic biodegradable and biocompatible material classi- NPs by high performance liquid chromatography (HPLC) method fied as GRAS (generally recognized as safe) and it is included in the FDA The entrapment efficiency of crocetin in crocetin-loaded NPs was inactive ingredients guide for formulating lipid based drug delivery assessed by reverse phase isocratic mode high performance liquid systems [25]. chromatography (RP-HPLC) (Waters 600, Waters Co., Milford, MA) Here, we have formulated crocetin-loaded lipid NPs to evaluate the followed by a previously described method [26]. The entrapment effi- antioxidant properties in a CsA-mediated toxicity in Human Embryonic ciency was calculated from the equation: Entrapment efficiency Kidney (HEK-293) cells in vitro. Wide spectrum of in vitro assay was (%) = (amount of crocetin in NPs/amount of crocetin used in for- conducted to study the antioxidant potential and protective role of mulation) × 100. crocetin in crocetin-loaded Nanoparticles (NPs) as compared to native counterpart. We have further studied the mechanism of protection by 2.4. In vitro free radical scavenging assay crocetin either in native or loaded in NPs by studying the expression of phase II detoxifying proteins (HO-1 expression) via Nrf2-mediated The 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging ac- regulation in in vitro. tivity was determined by following the protocol of Wu et al. [21]. The nitric oxide radical (NO%) scavenging activity was measured by fol- 2. Materials and methods lowing the protocol of Harput et al. [27]. The superoxide anion radical − (O2% ) scavenging activity was determined by following the protocol of 2.1. Materials Das et al. [28]. The percentages of scavenging of free radicals by the different Crocetin was purchased from MP, Biomedicals, Inc. (Germany). sample were calculated by the following formula: Polyvinyl alcohol (PVA, average MW = 31,000-50,000), 3-(4, 5-di- %of scavenging activity=− [(A A )/A )] × 100 methylthiazol 2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), di- 010 methylsulfoxide (DMSO), Pluronic F-127, Diphenyl picrylhydrazyle where A0 was the absorbance of the control and A1 was the absorbance