Cabazitaxel and thymoquinone co-loaded lipospheres as a synergistic combination for breast cancer

https://doi.org/10.1016/j.chemphyslip.2018.11.009Get rights and content

Highlights

  • Cabazitaxel(CBZ) as microtubule inhibitor and thymoquinone (TMQ) as HDAC inhibitor affects the important genes like p53, STAT3, Bax, BCL-2, p21 and down regulation of NF-κB are reported for potential activity against breast tumors.

  • However, poor aqueous solubility and permeability hinders the delivery of CBZ and TMQ to target site.

  • Developed CBZ and TMQ co-loaded lipospheres were characterized and evaluated for synergistic anticancer potential in breast cancer cell lines.

  • CBZ and TMQ co-loaded lipospheres have shown concentration dependent cell death and apoptosis due to rapid internalization into cells when compared to CBZ and TMQ combination solution.

  • Thus, as per observed results, it can be concluded that cabazitaxel and thymoquinone co-loaded lipospheres are the efficient delivery vehicles in management of breast cancer.

Abstract

Cabazitaxel as microtubule inhibitor and thymoquinone as HDAC inhibitor affects the important genes like p53, STAT3, Bax, BCL-2, p21 and down regulation of NF-κB are reported for potential activity against breast tumors. However, poor aqueous solubility and permeability hinders the delivery of these drugs to target site. To address the delivery challenges cabazitaxel and thymoquinone co-loaded lipospheres were developed. Lipospheres are the lipid based self-assemblies of particle size below 150 nm were prepared with more than 90% entrapment efficiency for both the drugs. In vitro drug release studies revealed there was a sustained diffusion controlled drug release from liposphere matrix leading to decrease in particle size with increase in zeta potential. Cytotoxicity studies on MCF-7 and MDA-MB-231 cells demonstrated cabazitaxel and thymoquinone as synergistic combination for the treatment of breast cancer which was proved by CompuSyn software. Enhanced efficacy of developed lipospheres can be due to rapid cellular internalization which was observed in confocal laser scanning microscopy. Drastic changes in cancer cell morphology such as nuclear fragmentation were observed upon treatment with these lipospheres in comparison to combination solution as observed in fluorescent imaging which are the hall marks of apoptosis. Cell cycle analysis and apoptosis studies confirmed the increased Sub G1 phase arrest as well as cell death due to apoptosis. Thus, as per observed results, it can be concluded that cabazitaxel and thymoquinone co-loaded lipospheres are the efficient delivery vehicles in management of breast cancer.

Introduction

Breast carcinoma is the leading cause of death in women in most of the developed countries (Stewart and Wild, 2017). The treatment remedies depend on the stage of the developed carcinoma. At early stages surgery is performed in combination with chemotherapy followed by radiation therapy. Late stages of breast carcinoma are difficult to cure and the life span of the patient will decline. Much of the chemotherapy is associated with poor absorption, penetration into tumor tissues which may lead to low therapeutic benefit, severe side effects and death in some cases (Miller et al., 2016). Combination therapy was emerged to improve the patient life expectancy by acting in either synergistic or additive manner or by reducing the side effects of the other chemotherapeutic (Pratt et al., 1994). However, lack of tumor targeting, toxicity to normal cells were the major drawbacks for available drugs. To overcome this situation, targeting the chemotherapeutics to disease site especially in case of carcinoma has been explored extensively nowadays (Ma et al., 2013).

Taxanes are the most promising microtubule inhibitors to eradicate several types of tumors (Jordan and Wilson, 2004). Drug resistance is the major hurdle which needs adjuvant therapy to suppress the disease progression and also to reduce the drug related side effects (Kitano, 2004). For reducing the resistance in monotherapy and to eradicate tumors, two or more chemotherapeutics are needed which will act by either synergistic or additive mechanism (Al-Lazikani et al., 2012). Cabazitaxel (CBZ) has been approved for hormone refractory prostate cancer. Jevtana® is the micellar CBZ solution with severe side effects like neutropenia, anaemia, thrombocytopenia, diarrhoea and neutropenic fever which leads to death limits its clinical use (Reddy and Bazile, 2014; Lydon, 2011). Recently CBZ has been reported for its activity in HER-2 negative breast cancer (Kümmel et al., 2017). Developing formulation for this drug is a challenging task as it possess poor aqueous solubility and permeability (Mahajan et al., 2015).

Thymoquinone (TMQ) is a phytochemical compound which has been extracted from the seeds of Nigella sativa/Black seed/Black cumin. TMQ exhibits anti-tumor activity against breast cancer (Pratama, 2017), ovarian cancer, lung cancer, colon cancer as well as in leukemia (Gali-Muhtasib et al., 2006; Khan et al., 2017). There are reports available that TMQ act as an angiogenesis inhibitor as well as an HDAC inhibitor, that it affects the important genes like p53, STAT3, Bax, BCL-2, p21 and cause down regulation of NF-κB (Kodappully Sivaraman Siveen et al., 2014; Rahmani et al., 2014) and also acts as better adjuvant therapy for cancer treatment (Mostofa et al., 2017). TMQ is a sensitive molecule which degrades in presence of light, heat, pH and also it has poor aqueous solubility which are the major drawbacks (Lebwohl and Ali, 2001; Salmani et al., 2014). Developing formulation for TMQ is a difficult task which can be overcome by loading in lipid based formulations.

Nanocarrier mediated delivery of chemotherapeutic agents with size less than 200 nm targets the tumor site either by passive manner (Enhanced permeation and retention (EPR) effect) or by active targeting (Surface functionalized nanocarriers with ligands/ aptamers/ antibodies). Biodegradable polymers and lipids are extensively used to deliver the drugs to targeted site (Muntimadugu et al., 2017; Peer et al., 2007). Lipids play a vital role in enhancing permeability through biological membranes of cells and tissues. Lipospheres (LSP) are the lipid based self-assembled systems containing solid hydrophobic lipid core surrounded by a layer of phospholipid molecules. LSP have several advantages over other lipid based systems such as low cost of reagents, ease of manufacturing, rapid dispersibility in an aqueous medium with controlled drug release rate. Cyclosporine loaded LSP (Deximune® soft-gelatin capsules Dexcel Pharma Ltd.) were in clinical use with enhanced oral bioavailability (Bekerman et al., 2004; Avramoff et al., 2012). LSP have been explored for topical delivery of chemotherapeutics (Avramoff et al., 2012; Jain et al., 2017, 2016) as they have capability to permeate the cells and tissues. But, very less reports were available to deliver these carriers loaded with drugs through parenteral administration (Elgart et al., 2012). In this study we investigated delivery of two drugs in LSP as a co-loaded nanocarrier for effective management of breast carcinoma synergistically with enhanced cellular permeation and cell death due to apoptosis.

Section snippets

Materials

CBZ obtained as a gift sample from TherDose Pharma Pvt. Ltd. (Hyderabad, India), Triolein (TO), Tripalmitin (TP), Trimyristin (TM) and N-methylpyrrolidone (NMP) were purchased from HiMedia Laboratories (Mumbai, India). Tricaprin (TC) and Tristearin (TS) were purchased from TCI chemicals Pvt. Ltd. (Tokyo, Japan). Thymoquinone, Egg phosphatidyl choline, Vitamin E-TPGS, Fluorescein isothiocyanate (FITC), Acridine orange, Ethidium bromide, Rhodamine, 3-(4, 5- dimethylthiazol-2-yl)-2,

Preparation and optimization of CBZ TMQ co-loaded LSP

Particle size and EE were considered as important parameters for optimization of LSP which will play crucial role in tumor penetration as well as to get therapeutic efficacy. CBZ TMQ co-loaded LSP were prepared by melt dispersion method. Optimization studies were performed initially for selection of solvent (NMP/ethanol) by keeping other parameters constant like type of core lipid (TC), coat lipid (Egg PC), surfactants (Tween 80, Vitamin E-TPGS) and their ratios (2:1:1:2) w/w respectively.

Conclusion

Co-delivery of synergistic drug combination from a single carrier system like LSP offers simultaneous delivery which will aid better therapeutic activity. Co-loaded LSP were developed for highly hydrophobic and unstable drugs like CBZ and TMQ with a particle size less than 150 nm which is more beneficial for passive targeting in tumors by EPR effect. CBZ and TMQ loading (for each drug with 4.268% w/w) were optimized with an entrapment efficiency of more than 90% for both the drugs. In vitro

Conflict of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Acknowledgement

Authors are thankful to NIPER-Hyderabad for providing required facilities, support and encouragement throughout the project. Authors thank Department of Biotechnology, Government of India (Grant no: 6242-P41/RGCB/PMD/DBT/WHDK/2015) for providing Cell culture facility. Authors are also grateful to TherDose Pharma Pvt. Ltd. for providing cabazitaxel and for research purpose.

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