*Article* **Co-Loaded Curcumin and Methotrexate Nanocapsules Enhance Cytotoxicity against Non-Small-Cell Lung Cancer Cells**

**Loanda Aparecida Cabral Rudnik 1, Paulo Vitor Farago 1,2, Jane Manfron Budel 1,\*, Amanda Lyra 1, Fernanda Malaquias Barboza 1, Traudi Klein 1, Carla Cristine Kanunfre 3, Jessica Mendes Nadal 1, Matheus Coelho Bandéca 4, Vijayasankar Raman 5, Andressa Novatski 1, Alessandro Dourado Loguércio <sup>1</sup> and Sandra Maria Warumby Zanin <sup>2</sup>**


Academic Editor: Maria Carla Marcotullio Received: 6 March 2020; Accepted: 17 April 2020; Published: 21 April 2020

**Abstract:** *Background*: As part of the efforts to find natural alternatives for cancer treatment and to overcome the barriers of cellular resistance to chemotherapeutic agents, polymeric nanocapsules containing curcumin and/or methotrexate were prepared by an interfacial deposition of preformed polymer method. *Methods*: Physicochemical properties, drug release experiments and in vitro cytotoxicity of these nanocapsules were performed against the Calu-3 lung cancer cell line. *Results*: The colloidal suspensions of nanocapsules showed suitable size (287 to 325 nm), negative charge (−33 to −41 mV) and high encapsulation efficiency (82.4 to 99.4%). Spherical particles at nanoscale dimensions were observed by scanning electron microscopy. X-ray diffraction analysis indicated that nanocapsules exhibited a non-crystalline pattern with a remarkable decrease of crystalline peaks of the raw materials. Fourier-transform infrared spectra demonstrated no chemical bond between the drug(s) and polymers. Drug release experiments evidenced a controlled release pattern with no burst effect for nanocapsules containing curcumin and/or methotrexate. The nanoformulation containing curcumin and methotrexate (NCUR/MTX-2) statistically decreased the cell viability of Calu-3. The fluorescence and morphological analyses presented a predominance of early apoptosis and late apoptosis as the main death mechanisms for Calu-3. *Conclusions*: Curcumin and methotrexate co-loaded nanocapsules can be further used as a novel therapeutic strategy for treating non-small-cell lung cancer.

**Keywords:** Calu-3 cell line; cancer chemotherapy; drug resistance; poly(ε-caprolactone); poly(ethylene glycol)

#### **1. Introduction**

Lung cancer is the deadliest of all cancers in the world. Sudden changes in lifestyle, environmental pollution, and smoking are strongly related to the development of lung cancer [1,2]. Treatment for lung cancer is typically guided by stage, although individual factors, such as overall health and coexisting medical conditions, are also important. Chemotherapy is beneficial at most stages of the disease, although it and radiation therapy are curative in only a minority of patients. The failure of chemotherapy in lung cancer treatment is mainly due to resistance mechanisms against chemotherapeutic agents, which result in a lack of therapeutic response [3,4]. Resistance phenomenon can occur by efflux pumps as P-glycoprotein (P-gp), which is expressed in human cells and acts as a localized drug transport mechanism, actively exporting drugs out of the cell [3,5,6]. In that sense, the efflux mechanism shows great clinical importance on lung cancer treatment and several compounds with P-gp inhibitor properties have been studied [7] to modulate chemotherapy resistance and to provide a more pronounced effect to chemotherapeutic drugs.

Curcumin (CUR) is a polyphenol obtained mainly from turmeric, the rhizome of *Curcuma longa* L. (Zingiberaceae), which is widely used in cooking as a spice and color additive due to its characteristic taste and deep yellow coloration [8,9]. CUR has demonstrated a variety of biological properties, such as antioxidant, anti-inflammatory, antimicrobial, and antitumor properties [8,10,11]. CUR has also shown therapeutic feasibility in improving wound healing activities by the use of nanotechnology-based delivery systems [11]. Moreover, CUR is a non-competitive inhibitor of P-gp by blocking the ATP hydrolysis process in efflux pump, which also paves the way for its use against lung cancer cells [7,12]. Despite these interesting effects, CUR has some disadvantages, such as its low aqueous solubility, low photostability, limited absorption, low bioavailability, rapid metabolism and elimination [13].

Methotrexate (MTX) is a well-known cytotoxic agent, which competitively and irreversibly inhibits dihydrofolate reductase, an enzyme that participates in tetrahydrofolate synthesis [14,15]. It is currently used in lung cancer treatment alone or along with other chemotherapeutic agents by oral and parenteral routes. High doses of MTX may circumvent at least two known mechanisms of resistance to this drug, membrane transport and high levels of the target enzyme. However, MTX can cause substantial toxicity by its systemic administration and can lead to remarkable side effects, such as hepatotoxicity, bone marrow depression, leucopenia, among others [16,17]. In that sense, the traditional use of this drug has two main problems when used against lung cancer: (a) its efflux from cancer cells by P-gp and (b) its high toxicity at usual therapeutic doses [18,19]. Thereby, new strategies to overcome these limitations are required.

Some controlled release strategies have been reported in the literature concerning the co-delivery of CUR and MTX. Dey et al. [20] developed gold nanoparticles containing CUR and MTX and evaluated their cytotoxic effect on C6 glioma cells and MCF-7 breast cancer cells. Curcio et al. (2018) [21] obtained pH-responsive polymersomes by self-assembling of a carboxyl-terminated PEG amphiphile achieved via esterification of PEG diacid with PEG40stearate. A highly hemocompatible co-delivery system of CUR and MTX was obtained. Vakilinezhad et al. [22] prepared PLGA nanoparticles for the co-administration of MTX and CUR as a potential breast cancer therapeutic system. Even though these authors reported a burst release from such nanoparticles, higher cytotoxicity was demonstrated against SK-Br-3 breast adenocarcinoma cell line. Curcio et al. [23] effectively delivered MTX to breast cancer cells by the use of a nanocarrier system derived from the self-assembly of a dextran-CUR conjugate prepared via enzyme chemistry with immobilized laccase acting as a solid biocatalyst. However, to the best of our knowledge, no previous paper was devoted to the preparation of co-loaded CUR and MTX nanocapsules using poly(ε-caprolactone) (PCL) as biodegradable polymer wall and poly(ethylene glycol) (PEG) as coating polymer focused on treating lung cancer.

Polymeric nanocapsules (NCs) are attractive colloidal systems to develop formulations containing labile and toxic substances. By definition, NCs are vesicular systems composed of a core, generally oily, surrounded by a polymer wall [24]. These carriers can present several advantages such as enhancing the dissolution process, increasing the therapeutic index, providing controlled delivery and achieving

protection from the photo and chemical degradation [25]. Therefore, NCs can circumvent limitations provided by both CUR and MTX since they allow drug protection against degradation, improve bioavailability, and reduce possible side effects. In particular, NCs can reach target tissues, certain affected organs, and tumors due to their superior features as small particle size, large surface area, Brownian motion, and surface functionality which could provide higher cytotoxic effect even at low doses of the chemotherapeutic agents [11,13]. Moreover, the two-drug combination into NCs can produce a higher objective response since CUR can potentiate MTX activity by delaying its efflux from the lung cancer cells.

Taking all these factors into consideration, this study was devoted to developing NCs for co-administration of CUR and MTX to provide a controlled release and a synergistic cytotoxic effect on non-small-cell lung cancer cell (Calu-3) growth. Moreover, in vitro studies were performed to evaluate the cytotoxic mechanism of these co-loaded NCs against Calu-3 cells.
