Incorporation of Zataria multiflora essential oil into chitosan biopolymer nanoparticles: A nanoemulsion based delivery system to improve the in-vitro efficacy, stability and anticancer activity of ZEO against breast cancer cells

Abstract

In the search of new alternative anticancer agents, essential oils (Eos) play a critical role, exerting selective anti-cancer properties and limiting the toxicity of conventional therapies. However, these compounds still face some challenges. Nanoemulsification (NE) protects labile and sensitive EO ingredients until they are released in the system. Herein, Zataria Multiflora Essential Oil (ZEO) loaded into chitosan (CS) nanoparticles was prepared in aqueous solution by mild emulsification into nanometric particles. FTIR spectroscopy exhibited no covalent interaction between active groups of ZEO and functional groups of CS. The outcomes revealed that CS/ZEONE increasingly improves the proliferation inhibition rate of Breast cancer cells as confirmed by MTT, morphological changes, DNA fragmentation and FACS analyses. Our findings suggested that CS/ZEONE exposure induces apoptosis, generates ROS, and triggers mitochondrial membrane permeabilization as well as DNA damage without harming normal cells. To find out the mechanism more precisely, the interaction of CS/ZEONE with gDNA was elucidated and Intercalative binding with strong stabilization of the DNA helix has been proposed. In conclusion, our data suggested that CS/ZEONE can be further explored as a promising antiproliferative and therapeutic candidate against breast cancer.

Introduction

Sustaining signals for continual proliferation along with growth suppressors evasion and circumventing cell death are the most basic traits of cancer cells [1,2]. Such traits can be considered as hotspots for targeting cancer cells by designing therapeutic strategies focused mainly on inducing apoptosis [1] and cellular arrest. Numerous naturally occurring substances with the ability to induce apoptosis in cancer cells have been suggested in the literature as potentials to prevent cancer or even as cancer therapeutic drugs [3,4]. Phytochemicals are generally considered innocuous and exhibit favorable effects against different types of diseases and may provide a safe alternative for cancer treatment [4,5]. Among phytochemicals, poly phenols that are found abundantly in essential oils have diverse biological activities and are widely used in medicine thanks to their biocidal (bactericidal, virucidal and fungicidal) and medicinal properties [[6], [7], [8]]. EOs have promising potentials for maintaining and promoting health, as well as preventing and potentially treating some diseases [6,9]. EOs possess antiproliferative, antimutagenic and detoxifying capabilities and act upon several pathways in cancer cells. Findings have demonstrated that EOs disrupt mitochondrial membrane potential causing ROS overproduction and reduction in cellular GSH (glutathione) level, release of Cytochrome C from mitochondria resulting in a disruption of Bcl2/Bax balance, upregulation of caspase 3 and caspase 9 activity, and PARP cleavage which collectively result in apoptosis. Moreover, EOs suppress mTOR (mechanistic target of rapamycin) and pPDK1 (protein pyruvate dehydrogenase kinase 1) triggering PKB dephosphorylation which dually deactivates mdm2 (murine double minute 2) leading to an increase in p21 to further initiate caspase activity and apoptosis. p21 upregulation also induces G1/S phase cell cycle arrest. In addition, EOs may decrease CDK7 which blocks CDK1/cyclin complex causing G2/M phase cell cycle arrest and subsequent apoptosis. Furthermore, EOs may directly inhibit mutagen entry into the cell or decrease Cyt C, thus preventing mutagen formation, and increase GST (glutathione S-transferase), UGT (uridine 5′-diphospho-glucuronosyltransferase), and EH (epoxide hydrolase) for enhanced detoxification [1].

Despite these outstanding biological activities, EOs utilization is very limited due to photosensitivity and rapid degradation [7]. Although attempts have been made to maintain the full potential of these oils, they are chemically labile and susceptible to oxidation and loss of unstable volatile compounds, particularly when exposed to light, oxygen, moisture, and heat [10]. In this regard, one way to overcome such limitations is to use carrier protection systems initially developed for health applications and are widely used in association with drugs, which enable modulation of the release profile.

Up to now, a variety of nano-sized drug carrier systems have been developed, among these, polymeric nanoparticles have received attention for biomedical purposes, which protect the bioactive compound from degradation and increase the distribution of drugs in cancer cells and reduce their toxicity toward normal cells/tissues [11,12]. The advantages of polymeric nanoparticles include biocompatibility, biodegradability, the ability to modify and functionalize the surface, incorporation of the active agent without any chemical reactions, and the possibility of modulating the degradation and release of the active agent [7,[12], [13], [14], [15]].

In a recent study, we found that ZEO, an EO extracted from zataria multiflora possesses antiproliferative properties against breast cancer cells and it might have potential uses in the field of cancer therapy. Notably, we found that ZEO can interact with genomic DNA, cause DNA strands breaks and DNA oxidation without any significant side effects on normal cells [16]. Given this background, in this study, we attempted to improve anti-cancer activity of ZEO using CS nanoparticles without causing substantial untoward effects on normal cells. A significantly large part of the current study on the emulsification of ZEO deals with nanometric size emulsion, which is used for the protection of the active compounds against environmental factors, to decrease oil volatility. Emulsification in nanometric particles is an alternative for overcoming these problems. Studies are ongoing in order to synthesis effective, water-soluble ZEO to enhance its anti-cancer potential and we provide further insight into the potential of this water-soluble formulation to enhance the anti-tumor activity of pure ZEO. With this study objective, we prepared CS/ZEONE dispersions by incorporation different concentrations of ZEO into CS solutions. After analyzing the particle size and Zeta-potential of CS and CS/ZEONE dispersions, we then tested the ability of CS/ZEONE to induce apoptosis in the human Breast adenocarcinoma cell lines, MDA-MB-231, T47D and MCF-7. Additionally, the drug was also concurrently examined on L929 normal fibroblast cells to reflect the drug's favorable selectivity toward cancer cells. In the past few years, there has been an increasing interest in the use of DNA as the key target for the development of effective natural and synthetic therapeutic agents in controlling DNA replication and gene expression [[17], [18], [19], [20]]. Therefore, investigating the interaction of CS/ZEONE with DNA may aid us to realize its metabolism and the mechanism of action at the molecular level. Small molecules bind to dsDNA primarily in three modes: (1) Electrostatic binding occurs along the negatively charged phosphate backbone of the DNA and cationic end of the ligands, (2) groove binding involving van der Waal's interaction in the deep major groove/ minor groove of the DNA helix, and (3) Intercalation binding describes agent intercalating into the adjacent bases of DNA [17,[20], [21], [22]]. Accordingly, one part of the study intends to confirm the binding of CS/ZEONE to DNA and the latter part reveals the mode of binding. Several analytical techniques were used to investigate this binding mechanism including fluorescence, UV absorption, and circular dichroism. All the experimental findings unambiguously establish that CS/ZEONE reside into the bases of the DNA helix. This study might provide valuable information on the interaction of CS/ZEONE with DNA and guide the design of more new binding reagents for DNA.