Elsevier

Carbohydrate Polymers

Volume 213, 1 June 2019, Pages 159-167
Carbohydrate Polymers

Chitosan nanogels as nanocarriers of polyoxometalates for breast cancer therapies

https://doi.org/10.1016/j.carbpol.2019.02.091Get rights and content

Highlights

  • Covalently crosslinked chitosan nanogels act as nanocarriers for Wells-Dawson POMs.

  • POMs show high encapsulation efficiency into chitosan nanogels.

  • POMs/chitosan nanogels display a pH-responsive release ability.

  • Chitosan nanogels enable significant and sustained release of Wells-Dawson POMs.

Abstract

Polyoxometalates (POMs) have been revealed as interesting antitumor agents inhibiting the action of Sox2 transcription factor, which reduces the risk of metastasis during hormonal therapies. However, they have shown serious concerns to be incorporated into the cells due to their cytotoxicity. Taking this into consideration, this study aims to develop polyoxometalate-based nanocarriers to be potentially applied as new forms of anticancer therapies. Thus, the Wells-Dawson type [P2Mo18O62]6− phosphomolybdate was physically loaded into covalently crosslinked chitosan nanogels that can act as nanocarriers for local delivery. The obtained nanocomposites were extensively characterized by 31P-NMR, TEM microscopy, DLS and zeta potential measurements. This work revealed that selected chitosan nanocarriers would present great potential for POM delivery into tumoral cells due to their pH-triggered deliverability that inhibits cytotoxic drug release at physiological pH. Furthermore, the high uptakes values reported herein make prepared nanocomposites interesting candidates for future breast antitumoral treatments.

Introduction

Cancer is one of the most serious health problems in our society. Among all the different types of cancer, breast cancer is the most frequent in western women (Soerjomataram, Louwman, Ribot, Roukema, & Coebergh, 2008). Since breast cancer typically overexpresses the receptor of estrogen (ER), hormone therapy by tamoxifen administration is the most common treatment for this disease. Unfortunately, tumors often develop resistance to hormone therapy, which has become a serious clinical problem. Indeed, currently, the lethality of cancer is based on the resistance to therapy, which leads to metastasis and subsequent recurrence (Piva et al., 2014). Thus, despite of the advances and efforts made in diagnosis and new therapies in the last years, there is still an urgent need for more sophisticated therapies, which implies the development of new materials. An effective approach to create advanced materials with biomedical purposes is the fabrication of hybrid systems composed of inorganic and organic components. Specifically, hybrid materials with stimulus responsive properties and colloidal size have emerged in the last years as intelligent nanostructures for controlled drug delivery and diagnosis (Cai, Zhang, Wei, & Cong, 2017).

Polyoxometalates (POMs) are discrete anionic metal-oxo clusters formed by early transition metals in their highest oxidation states (traditionally, V, Mo and W) (Pope & Müller, 1994). This family of compounds displays a unique compositional, structural and electronic variety that makes them suitable for applications in fields like catalysis, magnetism, energy and materials science (Sécheresse, 2013). Over the past decades, POMs have been identified as promising candidates for the development of different types of inorganic drugs, including antibacterial and antiviral agents (Bijelic, Aureliano, & Rompel, 2018; Shigeta, Mori, Yamase, Yamamoto, & Yamamoto, 2006) or remedies against Alzheimer’s disease or diabetes (Gao et al., 2014; Nomiya et al., 2001). Nevertheless, most of the biological studies have been devoted to their inherent anticancer activity, including human cancer lines (Xia et al., 2017; Yamase, 2005). It is worth highlighting the in vitro and in vivo activity of the isopropylammonium heptamolybdate (NH3iPr)6[Mo7O24]·3H2O and its photoreduction product against different cancer lines in mice. Their apoptotic growth suppression in tumors showed to be superior to that obtained for clinically approved drugs displaying good activity against breast, gastrointestinal, and intracranial cancer such as 5-fluorouracil and ACNU (Ogata et al., 2008; Yamase & Ishikawa, 2008). However, as indicated by the authors, the development of a drug-delivery system is required for its implementation as a novel anticancer drug with significant efficacy (Yamase, 2013). Some recent research has also shown that classical Wells-Dawson-type [P2Mo18O62]6− phosphomolybdates present an active role in breast cancer treatment, because they can inhibit the action of Sox2 (Narasimhan et al., 2011) and Ap-2 gamma (Hu et al., 2018) transcription factors that act as embryonic stem cell markers. Activation of both genes contributes to increase the resistance of the tumor cell to therapy and subsequently, tumor recurrence, limiting current hormonal treatments. For this reason, new therapies that incorporate Wells-Dawson-type POMs inhibitors could be more effective for the total elimination of breast cancer and prevention of future recurrence.

The interaction between POMs and biomacromolecules can be easily tuned in terms of polarity, electronic properties, shape, solution stability or acidity, by modifications in the cluster skeleton via compositional variations and organic functionalization (López, Carbó, Bo, & Poblet, 2012; Proust et al., 2012). Conversely, their lack of selectivity and cytotoxicity are one of the main drawbacks because they limit their use in biomedicine and usually make their insertion into cells very challenging. Thus, an interesting approach could be the incorporation of POM within biocompatible and non-toxic nanoreservoirs, which encapsulate them and ensure their specific release inside tumor cells.

In this sense, polymeric nanoparticles can act as nanocarriers of POMs isolating them from the surroundings, as exemplified by recent works on biocompatible starch-based systems (Wang, Liu, & Pope, 2003). Nanogels are polymeric hydrogels with nanometric size, which are dispersed as a colloidal solution. These are formed by polymer chains crosslinked by physical or covalent interactions and consequently, they swell in water, due to the presence of hydrophilic functional groups along the polymer chains, but maintaining their original shape (Dorwal, 2012). Based on their colloidal size, nanogels display interesting properties that make them suitable for biomedical applications, such as: i) low toxicity, ii) high efficiency entering and circulating through the body, or iii) easy functionalization (Chacko, Ventura, Zhuang, & Thayumanavan, 2012).

In addition, stimuli-sensitive nanogels could be specific enough to release loaded POM contents exclusively inside tumor cells. Among these stimuli, pH is frequently used in anticancer research, due to the difference in pH values between tumor extracellular environment (pH 6.5), or the acidic pH values of endosome and lysosome (5.0–5.5), in which nanogels can be internalized into the cells, and healthy tissues and bloodstream (pH 7.4) (Lee, Wang, & Low, 1996). Many investigations have been devoted to the study of pH-responsive nanogels for pH-triggered anticancer drug delivery along the last decades (Luan et al., 2017; Pérez-Alvarez, Sáez-Martínez, Hernaez, Herrero, & Katime, 2009; Yu, Hu, Pan, Yao, & Jiang, 2006). pH-sensitivity of these nanogels is usually based on the ionization at acidic pH of specific chemical groups from polymeric chains, such as primary amine derivatives. The resulting electrostatic repulsive forces lead to the swelling of the nanogels contributing to the controlled and specific drug loading and release. Thus, appropriate acid-swellable nanogels could transport POMs in the blood stream avoiding their damage as polymeric matrix remain collapsed at physiological pH, while loaded-POM content could be specifically delivered by diffusion or degradation (Shah et al., 2015) after nanogels are internalized into the cell.

Chitosan, poly[(1–4)-β-2-amino-2-deoxy-D-glucose], is a polysaccharide that is found naturally in the cell walls of some fungi, but is commercially obtained from the partial deacetylation of chitin. It is well known that chitosan exhibits excellent biocompatibility, biodegradability, non-toxicity, mucoadhesivity, and ability to open the tight junctions of cell membranes (Rinaudo, 2006). For all these reasons, it has been extensively used in the pharmaceutical and biomedical field. Drug delivery substrates based on chitosan have been prepared in a great variety of forms, like nanoparticles, microparticles, hydrogels, tablets, films, and fibers, for transdermal, nasal, ocular, oral, and parenteral administrations, among other applications (Shukla, Mishra, Arotiba, & Mamba, 2013). Furthermore, glucosamine units of chitosan ionize in dilute acidic solutions; therefore, chitosan behaves as a polycation, and crosslinked networks of chitosan show a pH-responsive swelling behavior suitable for specific loading and release of anticancer agents (Sahu et al., 2017). As a consequence, chitosan nanogels have been extensively studied in the last years to be applied in biomedicine, as drug delivery systems, especially with anticancer purposes (Pérez-Álvarez, Laza, & Álvarez-Bautista, 2016). Taking all this into account, this research aims to synthesize and characterize new hybrid nanodevices that could be interesting for future enhanced breast anticancer therapies. These nanocarriers were prepared by the successful encapsulation of Wells-Dawson-type P2Mo18 anions into water dispersible nanogels of chitosan.

The combination of chitosan and POMs is not a novel issue and has been addressed by other authors. POMs and chitosan/ carboxymethyl chitosan have been successfully assembled in form of macroscopic hydrogels (Azizullah, Haider, Kortz, Joshi, & Iqbal, 2017; Haider, Kortz, Ullah, & Sohail, 2017). In addition, a few attempts have been also made in the nanoscale. For instance, Geisberg et al. (Geisberger, Paulus, Carraro, Bonchio, & Patzke, 2011) studied the encapsulation of the [Co4(H2O)2(PW9O34)2]10− anion into negatively charged carboxymethyl chitosan matrixes (60–150 nm) formed by an extra ionic gelation with Ca2+, showing high encapsulation efficiency and reduced cytotoxicity. These authors also prepared POM-polymer composites by direct electrostatic interaction between [CoW11TiO40]8− species and cationic trimethylchitosan, showing superior POM uptake derived from attractive interactions between polyanions and chitosan chains (Geisberger, Gyenge, Maake, & Patzke, 2013). More recently, Shah et al. (Shah, Al-Oweini, Haider, Kortz, & Iqbal, 2014) employed also the direct assembly between chitosan and different Keggin, Anderson-Evans, Preysler and decavanadate anions to prepare nanocomposites that demonstrated a reasonable POM uptake of 86%. The same group also explored chitosan nanogels obtained by ionotropic gelation with tripolyphospate for encapsulation of three different derivatives of Keggin-type POMs, showing also high uptake efficiencies (Shah et al., 2015). However, to the best of our knowledge it is the first time that i) the encapsulation of the bioactive Wells-Dawson-type molybdate P2Mo18 has been studied; ii) POMs have been incorporated into covalently crosslinked chitosan nanogels, which are more stable than physical nanogels and iii) the effect of P2Mo18 loading into chitosan nanoparticles on the final particle size and zeta potential has been analyzed. This last point plays a crucial role in the design of the most effective action planning of P2Mo18-loaded chitosan nanogels for in vivo studies, because P2Mo18-chitosan interaction could modify the properties derived from the ionization of chitosan nanogels. For this reason, the uptake and release of POMs have been addressed on the basis of the pH-sensitive behaviour of nanogels in order to analyze if hybrid nano-assemblies are able to display selective POM release triggered by external pH changes.

Nanogels reported herein were prepared by covalent crosslinking of chitosan with poly(ethyleneglycol bis(carboxymethyl)ether), (PEG(COOH)2) (crosslinking degree: ˜23%), in reverse microemulsion medium and characterized through 1H-NMR spectroscopy. The morphology of the nanoparticles and the incorporation of POMs have been addressed by TEM microscopy, and 31P NMR spectroscopy. Besides, the zeta potentials and hydrodynamic diameters of unloaded and loaded nanogels were also measured as function of the external pH. Loading efficiencies of the Wells-Dawson anion were determined to be close to 90%. The release profile of the POM was studied and a controlled release under different pH conditions was observed. Obtained results enhance the potential use of the prepared nanogels for more effective therapies for the total eradication of breast tumors.

Section snippets

Materials

Chitosan (Aldrich, low molecular weight, Mw = 186,000 g mol−1 determined by GPC and DA = 21% determined by 1H NMR), poly(ethylene glycol)bis(carboxymethyl)ether, Mn  = 600 g/mol, (PEG(COOH)2), triton X-100, N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl carbodiimide hydrochloride) (EDC), sodium molybdate dihydrate (> 99.5%), potassium chloride (> 99%), phosphoric acid (85%), hydrochloric acid (37%) and hexanol (for synthesis, 98%) were purchased from Sigma-Aldrich. Cyclohexane

Preparation and characterization of P2Mo18 and chitosan nanogels

K6[α-P2Mo18O62]·12H2O yellow-greenish needle-like crystals were prepared as it is described in experimental part, and they were identified as a potassium salt of the well-known Wells-Dawson-type POM on the basis of infrared spectroscopy (FT-IR). FT-IR spectrum of P2Mo18 is depicted in Fig. 1. It is worth noting that the characteristic bands of strong intensity in the region below 1200 cm−1 are associated with plenary α-Wells-Dawson-type phosphomolybdates. The antisymmetric stretching

Conclusions

The present study demonstrates that covalently crosslinked chitosan nanogels are suitable nanocarriers to encapsulate bio-active Wells-Dawson type P2Mo18O62]6− anion. The successful POM encapsulation into chitosan nanogels could be demonstrated by 31P-NMR, TEM microscopy, DLS and zeta potential determination. The P2Mo18–chitosan nanocomposites displayed negative zeta potentials, larger particle sizes than the unloaded chitosan nanogels and diminished pH-responsive swelling, as a consequence of

Acknowledgments

This work was financially supported by the Spanish Ministry of MINECO (MAT2017-89553-P), the Government of Basque Country (Grupos de Investigación, IT718-13, ELKARTEK bG18 (KK2018/00054)) and UPV/EHU (PPG17/37; GIU17/050). Authors are also grateful to the technical and human support provided by SGIKER (UPV/ EHU, MICINN, GV/EJ, ERDF, and ESF).

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