CD44 receptor targeted ‘smart’ multi-walled carbon nanotubes for synergistic therapy of triple-negative breast cancer
Graphical abstract
Introduction
Triple-negative breast cancer (TNBC), lacks the presence of estrogen, progesterone, and human epidermal growth factor-2 (HER-2) receptors and therefore very challenging to diagnose and treat [1,2]. Besides, the low frequency of TNBC driven by rare aberrations imposed limitations for ad hoc drug development and lack of specific marker of the TNBC is another big challenge to design formulation strategy. TNBC treatment involves chemotherapy and radiation therapy, which suffers from severe drawbacks such as multidrug resistance, cytotoxicity, and tissue damage. Hormonal therapeutics is not recommended treatment for TNBC due to the loss of target receptors. Till date, surgery and chemotherapy, individually or in combination, are the only available treatment for TNBC [3,4].
Moreover, the majority of chemo-specific molecules fails to attain effective therapeutic concentration without harming the normal cells [5], due to their nonselective tumor accumulation [6]. Effective and safe treatment of TNBC requires the maximum exposure of therapeutic molecules to cancerous tissues/cells while minimum reachability to non-cancerous cells, and that could be achieved by selecting an appropriate targeted drug delivery system [[7], [8], [9]].
Nanotechnology-based approaches for delivering therapeutic cargo could serve as an effective strategy to overcome most of the adverse effects resulting from the use of conventional chemotherapy practice [10,11]. Reports are available suggesting that nanotechnology-based designer delivery systems such as dendrimers, liposomes, polymeric nanoparticles, and carbonaceous materials have improved anticancer efficacy and redefined the way of delivering the drugs to the cancer sites by imparting a high level of specificity [[12], [13], [14]]. Among them, multi-walled carbon nanotubes (MWCNTs) have gained wide attention in engaging the therapeutic molecules for effective and potential treatment of various cancers including breast, lung and liver cancer owing to their excellent physicochemical properties [15]. Our selection of MWCNTs over SWCNTs was due to high drug entrapment efficiency inside the cone and inner walls of MWCNTs along with minimum drug leakage over time and excellent mechanical strength than that of SWCNTs. Besides, large scale production of MWCNTs is much cheaper than that of SWCNTs [[15], [16], [17]]. Recently, Arkan et al., employed gold nanoparticles modified MWCNTs to fabricate novel immunosensor for the detection of HER2 in serum samples as a lead in breast cancer treatment [18]. Similarly, Prajapati et al., evaluated in vitro and in vivo potential of gemcitabine (GEM) loaded hyaluronic acid (HA) conjugated multi-walled carbon nanotubes (GEM/HA- PEG-MWCNTs) for effective colon cancer targeting [19]. However, to obtain targeted/site-specific delivery, accurate placement and accumulation of drug molecule are required in the cancer cells, which can be made possible by increasing the cellular uptake of the drug via employing suitable targeting ligand.
In the past couple of years, Hyaluronic acid (HA), as naturally occurring carbohydrate (glycosaminoglycan) based multiunit chain of d-glucuronic acid and glucosamine, has been widely explored as targeting tool owing to its high specificity towards CD44 receptors overexpressing cancer cells [[20], [21], [22]]. As a fact, CD44 receptors are highly expressed on the surface of a variety of cancer cells and reported to plays a very crucial role in cellular responses such as cellular proliferation, migration, and invasion [[23], [24], [25]]. Another aspect of using HA as targeting ligand is the availability of functional groups on its surface that could be engineered to form specific conjugates for particular applications [26]. Yao et al. recently developed SWCNTs as drug delivery platforms and achieved higher intracellular epirubicin concentration to overcome multidrug resistance in A549/Taxol cells [27]. Similarly, Cao et al., fabricated MWCNTs functionalized using HA for the treatment of breast cancer [28]. However, the synergistic anti-cancer therapy is the need of the hour and addition of specific molecules that provoke the anti-cancer effect in cancer cells while un-affecting the healthy cells is more promising.
In context, α-Tocopheryl succinate (α-TOS; vitamin E succinate) which is a succinyl derivative of vitamin E, well-known for its anti-cancer properties mainly via an apoptotic pathway and at the same time reported being non-toxic to healthy cells [29]. This property makes α-TOS particularly crucial in achieving synergistic anticancer effects along with the other therapeutic molecules. In a recently published literature, investigators found that α-TOS enhances the anti-tumor activity of pterostilbene in human breast cancer cells in vivo and in vitro. Pterostilbene (dietary supplement) in association with α-TOS found to synergistically maximize the cytotoxicity to breast cancer cells, by disrupting signal transduction, transcription factors, and cell cycle proteins [30].
The present investigation was aimed to design the combined, synergistic and targeted therapy as a novel treatment strategy for TNBC by utilizing the HA (as CD44 receptor targeting ligand), α-TOS (to produce a synergistic anticancer effect) and Dox (as chemotherapeutic agent) delivered via MWCNTs as drug delivery platform. This strangely will be beneficial for the treatment of TNBC over the alone chemotherapy or surgery or a combination of both. The core objective behind the development of multifunctional MWCNTs was to achieve maximum therapeutic effects in MDA-MB-231 cells while reducing the associated side effect by chemotherapy alone. In a step by step synthesis, pristine MWCNTs were converted to aminated MWCNTs following successful carboxylation and acylation in different stages. Afterward, aminated MWCNTs were reacted with α-TOS-PEG-COOH and HA-PEG-COOH which were prepared by reacting α-TOS and HA with NH2-PEG-COOH, using well-known NHS-EDC coupling chemistry in a separate reactions. Dox was loaded into finally obtained α-TOS-HA-MWCNTs conjugate to get α-TOS-HA-MWCNTs/Dox. The modified MWCNTs, as obtained from different steps, were characterized for their size, polydispersity index (PDI), surface charge, and surface morphology. Drug release was determined in acidic pH (5.0) in addition to normal physiologic pH (pH 7.4). The anti-cancer potential was assessed using the sulforhodamine B (SRB) assay and apoptotic assay. Cellular uptake of α-TOS-HA-MWCNTs was compared with non-HA functionalized formulations to determine the targeting efficiency of HA. Besides, hemocompatibility of different modified MWCNTs formulations were determined to be used as parenteral formulation.
Section snippets
General experimental materials and cell culture materials
Doxorubicin Hydrochloride (Dox) was obtained as gift from Sun Pharmaceuticals, Vadodara, India. Pristine multi-walled carbon nanotubes (Pristine MWCNTs; Purity >98%, length 3-8 μm, and diameter 10–20 nm), α-Tocopheryl succinate (α-TOS) and Poly (Ethylene Glycol)- 2- Amino Ethyl Ether Acetic Acid (COOH-PEG-NH2) (MW 2100 Da) were procured from Sigma Aldrich, India. Polytetrafluoroethylene (PTFE) filters (0.22 μm and 0.45 μm pore size) were purchased from Rankem, India. Hyaluronic acid (HA) and
Carboxylation, acylation and amination of MWCNTs
Efficient, safe and targeted therapy is of paramount importance in treating TNBC and the current focus area for the development of the majority of nanomedicines for TNBC treatment [38]. In the last couple of years, among the carbon-based materials for delivery of small therapeutic molecules, MWCNTs have explored extensively owing to their excellent properties such as high drug loading and mechanical strength. MWCNTs are widely reported to be used in nanotechnology for theragnostic applications.
Conclusion
In this investigation, the CD44 receptor-targeted α-TOS and HA tailored and Dox loaded MWCNTs (α-TOS-HA-MWCNTs/Dox) with synergistic anticancer formulation was successfully prepared and characterized to serve against TNBC. Dox was utilized as a model anticancer therapeutic molecule for effective treatment. The strategy was to combine the anticancer potential of Dox, targeting potential of HA and synergistic effect of α-TOS to treat an aggressive form of breast cancer, TNBC. The in vitro
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.
Acknowledgements
Authors are deeply grateful to the Department of Science and Technology (DST), for providing research fund under women scientist program (SR/WOS-A/LS-314/2016) to Ms. Nidhi Jain Singhai. Authors would also thankful to the Rajiv Gandhi Technological University for providing necessary infrastructure and facility to carry out the experiments.
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