Research paper
pH-Dependent doxorubicin release from terpolymer of starch, polymethacrylic acid and polysorbate 80 nanoparticles for overcoming multi-drug resistance in human breast cancer cells

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Abstract

This work investigated the capability of a new nanoparticulate system, based on terpolymer of starch, polymethacrylic acid and polysorbate 80, to load and release doxorubicin (Dox) as a function of pH and to evaluate the anticancer activity of Dox-loaded nanoparticles (Dox-NPs) to overcome multidrug resistance (MDR) in human breast cancer cells in vitro. The Dox-NPs were characterized by Fourier transform infrared spectroscopy (FTIR), isothermal titration calorimetry (ITC), transmission electron microscopy (TEM), and dynamic light scattering (DLS). The cellular uptake and cytotoxicity of the Dox-loaded nanoparticles were investigated using fluorescence microscopy, flow cytometry, and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay. The nanoparticles were able to load up to 49.7 ± 0.3% of Dox with a high loading efficiency of 99.9 ± 0.1%, while maintaining good colloidal stability. The nanoparticles released Dox at a higher rate at acidic pH attributable to weaker Dox–polymer molecular interactions evidenced by ITC. The Dox-NPs were taken up by the cancer cells in vitro and significantly enhanced the cytotoxicity of Dox against human MDR1 cells with up to a 20-fold decrease in the IC50 values. The results suggest that the new terpolymeric nanoparticles are a promising vehicle for the controlled delivery of Dox for treatment of drug resistant breast cancer.

Graphical abstract

Possible mechanism of MDR reversal by doxorubicin loaded PMAA–PS 80-g-St nanoparticles, exhibiting pH-dependent doxorubicin release, in Pgp-over expressing human breast cancer cells.

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Introduction

Two major limitations of traditional chemotherapy are dose-limiting systemic toxicity and the development of multi-drug resistant (MDR) phenotypes. Traditional chemotherapy is not specific to cancer cells. In most cases, both cancerous and healthy cells are exposed to anticancer drugs. Chemotherapeutic agents such as doxorubicin (Dox) are more toxic toward actively proliferating cells [1]. However, like cancer cells, many normal cells, for example, gastrointestinal tract, hair follicle, and bone marrow cells, are continually proliferating, making them susceptible to the toxicity of chemotherapeutics. Moreover, many anticancer drugs produce reactive oxygen species through redox cycling reactions, which are toxic to both cancerous and normal cells [2].

Another major barrier to effective cancer chemotherapy is drug resistance of cancer cells [3], [4]. This drug resistance phenotype can be acquired following failed rounds of chemotherapy, or it can be innate and pre-programmed into the physiome of the cancer cells [3]. Moreover, the resistance phenotype is rarely specific to one drug, but rather manifests itself as a cross-resistance to an array of drugs with different chemical structures [5]. This cross-resistance phenotype is often attributed to cell membrane drug transport proteins, metabolic pathways, and intracellular targets common to cancer chemotherapeutics and is known as MDR [3], [4], [5], [6].

Dox, a well known anticancer drug, is a member of the anthracycline ring antibiotics with a broad spectrum of antitumor activity against a variety of human and animal solid tumors [7], [8]. The drug, however, has a very narrow therapeutic index and its clinical use is hampered by several undesirable side effects including cardiotoxicity and myelosuppression [7], [8], [9], [10]. Another drawback of Dox is that it is a substrate of multiple membrane efflux transporters such as p-glycoprotein (P-gp), multidrug resistance protein 1 (MRP1), and breast cancer resistance protein (BCRP) [11], [12]. These transporters prevent intracellular accumulation of many anticancer agents causing a reduction in their cytotoxicity. P-gp mainly prevents active uptake and increases cellular efflux of positively charged amphipathic drugs such as Dox in an ATP-dependent manner. In fact, P-gp over-expression is suggested as one of the main mechanisms of MDR in cancer cells [3], [6], [11], [12].

Encapsulation of Dox in nanoparticles has been found to enhance its cytotoxicity by circumventing membrane transporter-mediated MDR in cancer cells [13], [14], [15], [16], [17]. The nanoparticles carry the drug into cancer cells mostly by endocytosis, transporting the drug to the cytoplasm or perinuclear region and releasing the drug inside the cells [15], [16], [18], [19]. Drug molecules are not exposed to the binding sites of trans-membrane efflux pumps, for example, P-gp, and are less likely to be pumped out of the cancer cells.

It has been observed that nanoparticles can accumulate in many solid tumors at much higher concentrations than in normal tissues or organs by a nonspecific targeting process known as the enhanced permeation and retention (EPR) effect [20], [21]. The EPR effect is attributed to leaky vasculature and limited lymphatic drainage, typically found in solid tumors. Ideally, the cytotoxic drugs should be released from the nanocarriers within the tumor tissue or inside the tumor cells after uptake by active or passive transport mechanisms. Hence, a nanoparticulate system with minimal drug release in the systemic circulation but accelerated release once in the tumor tissue is highly desirable. One way to achieve this goal is to take advantage of the enhanced acidic environment of tumor tissue and cellular endosomes/lysosomes. It is well documented that tumor extracellular pH is lower than that in normal tissue with average pH values of 6.9 to as low as 5.7 owing to different metabolic pathways in cancer cells [22], [23]. To take advantage of this pathophysiological characteristic of the tumor microenvironment, polymeric nanoparticles with pH-dependent drug release properties have been studied by various research groups [24], [25], [26], [27], [28]. These systems often change their physical and chemical properties, such as swelling ratio or solubility in response to local pH level leading to an increase in drug release rate at lower pH. Alternatively, drugs have been conjugated to the carrier system through an acid labile bond which promotes accelerated release under mildly acidic conditions [28], [29].

Recently, we have developed a new nanoparticulate system based on the terpolymer of poly(methacrylic acid)–polysorbate 80-grafted-starch (PMAA–PS 80-g-St) that exhibits pH-sensitivity due to the presence of PMAA [30]. In this terpolymer system, starch, PS 80, and PMAA (pKa = 5.6–7) are commonly used ingredients in various pharmaceutical formulations approved by regulatory agencies for use in humans. Starch is a food ingredient degradable by amylase in the body, and hence, this nanoparticle system is expected to possess good biocompatibility and biodegradability properties. Moreover, although not the focus of this study, polysaccharide coatings have recently been considered as an alternative to PEG coatings for giving “stealth” properties to nanoparticles [31]. Certain polysaccharides such as heparin, dextran, and hydroxyethyl starch have been shown to prolong the nanoparticle circulation in blood and minimize their interaction with blood proteins and phagocytotic cells [32], [33], [34]. The carboxyl groups in PMAA are expected to provide binding sites for cationic anticancer drugs such as Dox as suggested in previous studies using other carboxyl-containing and negatively charged polymers [15], [17].

Polysorbate 80 (PS 80) is a polyethylene sorbitol ester with a molecular weight of 1310 Da. It is widely used as an emulsifier/surfactant/stabilizer in pharmaceutical formulations. The surfactant has a sorbitan ring with ethylene oxide polymers attached at three different hydroxyl positions and contains mixture of fatty acid side chains which are attached through an ester linkage to the ethylene oxide oxygen. The major fatty acid component of the side chain is oleic acid contributing to more than 60% of the total composition of the side chain.

Herein, we investigated the ability of PMAA–PS 80-g-St nanoparticles to efficiently load and release Dox and characterized the molecular interactions of Dox with the polymer particle as a function of pH. We also studied cellular uptake of the nanoparticles and the efficacy of Dox-loaded nanoparticles in overcoming MDR in human breast cancer cells in vitro.

Section snippets

Chemicals

Soluble corn starch (MW = 11,000 g/mol), methacrylic acid (MAA), N,N′-methylenebisacrylamide (MBA), sodium thiosulfate (STS), potassium persulfate (KPS), polysorbate 80 (PS 80), and sodium dodecyl sulfate (SDS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), fluoresceinamine isomer I (FA), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and all other chemicals, unless otherwise mentioned, were purchased from Sigma–Aldrich Canada

Properties of PMAA–PS 80-g-St nanoparticles and their high capability of efficiently loading Dox without loss of colloidal stability

Table 1 summarizes the Dox loading content, loading efficiency, particle size, and ζ-potential for nanoparticles with different Dox/COOH molar ratios. The particle size ranges from 184 to 230 nm with the polydispersity index (PdI) of 0.09–0.12. Fig. 1A presents a typical particle size distribution plot of Dox-loaded PMAA–PS 80-g-St nanoparticles (Dox-NPs) (LC = 33%) in pH 7.4 PBS measured by DLS. TEM of Dox-NPs shows a nearly spherical shape and porous morphology with the average diameter of about

Discussion

The starch, PMAA, and PS 80 terpolymer nanoparticles were prepared by a one-pot aqueous polymerization method which enables simultaneous starch grafting and nanoparticle formation eliminating the need for use of oils and/or organic systems [30]. This fabrication method has advantages over other previously described polysaccharide based nanoparticle fabrication methods. The mechanism of nanoparticle formation during polymerization is similar to that for making N-isopropylacrylamide polymers and

Conclusions

The new PMAA–PS 80-g-St nanoparticles are able to effectively load high quantities of Dox without noticeable loss of their colloidal stability. The particles provide pH-dependent release of the drug in vitro with the release rate being significantly higher at mildly acidic pH than at neutral pH imparting differential drug release between tumor tissue and normal tissue. Dox-NPs can overcome P-gp MDR1 mediated MDR in human breast cancer cells, significantly improving the cell killing efficacy of

Acknowledgements

The authors sincerely acknowledge partial financial support from BioPotato network (Co-leaders: Drs. Helen Tai and Yvan Pelletier), Agricultural Bioproducts Innovation Program (ABIP) of Agriculture & Agri-Food Canada; NSERC Discovery grant and equipment grant, and CIHR equipment grant to X.Y. Wu, Ontario Graduate Scholarship and Ben Cohen top up award to A. Shalviri, NanoNetwork award to G. Ravel, and technical assistance in the measurement and analysis TEM data from Dr. Battista Calvieri.

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