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
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.
Section snippets
Particle size and zeta potential measurements
Nanoemulsification of EOs represents a feasible and efficient approach to modulate drug release, increase the physical stability of the active substances, protect them from the interactions with the environment, decrease their volatility, enhance their bioactivity, reduce toxicity, and improve patient compliance and convenience [9].
Zeta potential is an important marker of the stability of various colloidal dispersions. The nanocarriers with particle size of 50–300 nm, positive zeta potential
Conclusion
Fabrication of a novel formulated therapeutic agent for enhancing the apoptotic potential in invasive MDA-MB-231 Breast cancer cells with minimal effect on normal cells was the main goal in the present study. DLS, zeta potential and FTIR analysis all support the successful surface functionalization of ZEO by CS NPs. Based on the findings, we conclude that CS/ZEONE principally induces the production of ROS inside the mitochondria of MDA-MB-231 cells, which provokes many cellular events leading
Preparation of CS/ZEO dispersion
Chitosan was purchased from Sigma chemical Co and CS stock solution (1mgml−1) was prepared as described previously [43]. ZEO was a well-defined complex mixture of aromatic compounds and was obtained as describe previously [16]. The ZEO-in-water nanoemulsions were prepared by mild emulsification conditions. For the preparation of CS/ZEO dispersions, ZEO (100 mg/100 mL of CS) was added to the dispersions and mixed under mechanical stirring at 800 RPM for 12 h at 40 °C. The emulsifier tween-20
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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