Elsevier

Carbohydrate Polymers

Volume 198, 15 October 2018, Pages 142-154
Carbohydrate Polymers

TPGS-functionalized and ortho ester-crosslinked dextran nanogels for enhanced cytotoxicity on multidrug resistant tumor cells

https://doi.org/10.1016/j.carbpol.2018.06.079Get rights and content

Highlights

  • TPGS was grafted on dextran nanogels to inhibit the drug efflux activity of P-gp.

  • Ortho ester bonds endowed the nanogels’ pH-degradable performance.

  • These Nanogels performed pH-triggered drug release in mildly acidic conditions.

  • TPGS-grafted nanogels could significantly reverse DOX-resistance on MCF-7/ADR cells.

  • TPGS-grafted nanogels possessed strong lethality against MCF-7/ADR tumor spheroids.

Abstract

Herein pH-sensitive nanogels (NG1) and P-glycoprotein-repressive nanogels (NG2) were prepared by copolymerization between an ortho ester crosslinker (OEAM) and tocopheryl polyethylene glycol succinate (TPGS)-free or conjugated dextran. Nanogels with or without TPGS possessed a uniform diameter (∼180 nm) and excellent stability in various physiological environments. Doxorubicin (DOX) was successfully loaded into NG1 and NG2 to give NG1/DOX and NG2/DOX, both of them showed appropriate drug release profiles under mildly acidic conditions (pH 5.0). NG2/DOX possessed higher drug enrichment and lethality than NG1/DOX did on MCF-7/ADR cells. Analysis of corresponding index of efflux activity showed that NG2 could induce depolarization of mitochondrial membrane and interfere with ATP metabolism. NG2/DOX also displayed increased penetration and growth inhibition on MCF-7/ADR multicellular spheroids. These results demonstrated that pH-sensitive TPGS-functionalized nanogels (NG2) as drug carriers had great potential to suppress drug efflux in MCF-7/ADR cells and even overcome MDR on cancer cells.

Introduction

Multidrug resistance (MDR) has been observed on a variety of mechanistically and structurally unrelated anticancer drugs, and always compromises the chemotherapeutic effect in clinical trials (Dubey et al., 2016; Johnson & Chen, 2017; Krishna & Mayer, 2000). Recently, a large number of research has shown that MDR is associated directly with the overexpression of P-glycoprotein (P-gp), a transporter of exogenous substrates (anticancer agents) on cell membrane related to drug efflux (Braunová et al., 2017; Callies et al., 2016; Holohan, Van Schaeybroeck, Longley, & Johnston, 2013; Sun et al., 2004). Drug delivery systems based on inhibition of efflux have been widely utilized trying to overcome MDR, but no significant progress has been achieved due to undesirable side effects and inadequate curative performance (Bao et al., 2014; Bernabeu et al., 2014; Kapse-Mistry, Govender, Srivastava, & Yergeri, 2014; Li et al., 2016; Muthu, Kutty, Luo, Xie, & Feng, 2015). Therefore, it is urgent to develop a particular drug delivery system to enhance drug enrichment in MDR cells and inhibit drug efflux with minimal side toxicity (Koziolová et al., 2016).

As a commercially available polysaccharide, dextran (DEX) has been widely used to prepare drug delivery carriers because of its excellent biocompatibility, biodegradability, non-toxicity as well as easiness for modification (Du, Weng, Yuan, & Hu, 2010; Kaewprapan, Inprakhon, Marie, & Durand, 2012; Sun et al., 2010). Besides, DEX possesses excellent aqueous solubility, non-fouling and non-ionic properties, consequently generating a super stability in blood circulation (Bai et al., 2018; Kreuter, 1994, ch. 5; Su, Jia, & Shan, 2016). Fréchet et al. had successfully prepared a novel type of nanoparticles based on acetalated-dextran, and results indicated that their delivery system possessed a super-biocompatibility similar to the FDA approved material, poly (lactic-co-glycolic acid) (Bachelder, Beaudette, Broaders, Dashe, & Fréchet, 2008). Sagnella et al. proved that dextran-based nanocarrier possessed a strongly penetrating ability into 3D tumor spheroids (Sagnella et al., 2013). In addition, biodegradable dextran-based nanogels had been fabricated by Thienen et al., which showed excellent stability and wouldn’t aggregate after incubation with human serum in vitro (Van Thienen, Raemdonck, Demeester, & De Smedt, 2007). Although these DEX-based carriers have such excellent physiology compatible properties, formulations from simply modified-dextran have failed to effectively inhibit the efflux of MDR cells, and it is necessary to incorporate a reasonable and appropriate functionalization into dextran to combat with MDR.

Tocopheryl polyethylene glycol succinate (TPGS) is a non-ionic water-soluble compound formed by vitamin E and polyethylene glycol (PEG), as such it inherits advantages form these two materials, such as being amphiphilic as well as retaining benefits of PEGylation (Zhang, Tan, & Feng, 2012). TPGS has been used as emulsifier, solubilizer, absorption enhancer, and permeation enhancer based on the above excellent properties (Sadoqi, Lau-Cam, & Wu, 2009). More importantly, TPGS has also been extensively applied to combat MDR cancer because it can effectively interfere with cell membrane fluidity, and then, drug efflux is inhibited by interfering with energy metabolism and P-gp transport function (Collnot et al., 2010; Gottesman, Fojo, & Bates, 2002; Guo, Luo, Tan, Otieno, & Zhang, 2013; Silva et al., 2015). For instance, Zhu et al. prepared a series of PLGA nanoparticles encapsulated with various content of TPGS, and results indicated that TPGS-associated nanocarriers could prolong drug retention time and increase drug concentration by synergistically targeting mitochondria to decrease the mitochondrial membrane potential (MMP) (Zhu et al., 2014). Nevertheless, these P-gp inhibitors-contained carriers often caused undesirable off target toxicity and failed to quickly reach the cytotoxic threshold of anticancer drugs (Bernabeu et al., 2014; Ferry, Traunecker, & Kerr, 1996; Yu et al., 2015). Consequently, an accurate spatial-temporal drug release performance is highly required for TPGS-contained carriers to effectively induce MDR cells apoptosis.

Acid-triggered degradation is widely applied in controlled drug release, owing to the distinctive pH gradient among blood vessels (pH 7.4), extracellular (pH 6.5–7.2), and intracellular (pH 5.0–6.0) space in solid tumors (Lee, Gao, & Bae, 2008; Luo, Sun, Sun, & He, 2014). Hence, combining P-gp resistant molecules and pH-sensitive components is an effective method to combat MDR in cancer (Wang et al., 2011). Guo et al. fabricated pH-sensitive and TPGS-grafted chitosan nanoparticles to overcome the P-gp-induced MDR, and corresponding results showed that intracellular drug level significantly improved after treatment by nanoparticles with different TPGS grafting degree (Guo, Chu et al., 2013). Unfortunately, lack of acid-sensitivity of the above TPGS-contained delivery system limited its anti-cancer effect, only about 25% of DOX was released from nanoparticles after incubation at pH 5.5 even for 7 d. In the past several decades, ortho ester has been widely reported as an acid-sensitive linkage with great potential applications (Fu et al., 2017). Compared with other acid-labile bonds such as ketal and acetal, the hydrolysis rate of ortho ester could be increased by 1 to 4 orders of magnitude under acidic conditions (Yan et al., 2017). Recently, we reported an ortho ester-based compound (OEAM) as a cross-linker to prepare pH-sensitive nanogels. These carriers were highly stable under physiological condition while specifically showed swelling and dissociation in mildly acidic environments (such as pH 5.5) (Yang et al., 2017; Zha et al., 2017). Subsequently, encapsulated drugs were controllably enriched in cancer cells and quickly reached their effective cytotoxic threshold for enhanced cytotoxicity.

We hypothesized a desirable drug carrier for MDR cancer therapy, which could be realized by integrating TPGS and ortho ester-bonds into dextran nanogels to achieve an ideal delivery process: (i) remaining stable and long-circulating in blood vessels; (ii) triggering encapsulated drug release at tumoral intracellular pH; (iii) inhibiting cell efflux to prolong the retention time of anticancer agent and quickly reaching an effective cytotoxic threshold in MDR cells, and then to obtain enhanced cytotoxicity.

In this work, we prepared the proposed TPGS-grafted dextran nanogels crosslinked by OEAM. The structures of modified-dextran and physicochemical properties of these nanogels were investigated in details. The MDR-related analysis such as the change of ATP metabolism and mitochondrial transmembrane potential, were performed in vitro by using a MCF-7/ADR monolayer cell model and three-dimensional multicellular spheroids.

Section snippets

Materials

Dextran (20 KDa, α-(1–3) branch, 5%), 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and poly (2-hydroxyenthyl methacrylate) (poly-HEMA) were purchased from Sigma-Aldrich (St Louis, MO, USA). N,N'-Carbonyldiimidazole (CDI) was gained by Tokyo Chemical Industry Co., Ltd (Shanghai, China). Methacrylic anhydride (MA), potassium peroxodisulfate (KPS, re-purified by distill waster) and TPGS were obtained from Shanghai Macklin Biochemical Co., Ltd. Dimethyl sulfoxide (DMSO) was

Synthesis and analysis of MA-dextran and TPGS-MA-dextran

The synthetic routes of dextran derivatives were shown Fig. 1. Methacrylic anhydride was grafted onto dextran (MA-DEX) by the substitution reaction between estolide and hydroxyl (Fig. 1a). In order to prepare TPGS grafted MA-DEX (TPGS-MA-DEX), TPGS was first activated by N,N'-Carbonyldiimidazole to convert to CDI-TPGS (Fig. 1b). And then, TPGS-MA-DEX was achieved by mixing CDI-TPGS and MA-DEX at a certain molar ratio (Fig. 1c).

Besides, the chemical structures of MA-DEX and TPGS-MA-DEX were

Conclusions

In summary, pH-sensitive dextran-based nanogels with or without TPGS-functionalization were prepared to overcome DOX efflux in multidrug resistance breast cancer cells. Both nanogels with or without TPGS displayed an excellent stability in various physiological conditions on account of the chemical cross-linking between ortho ester and dextran derivatives. On the contrary, these nanogels swelled and degraded in mildly acidic environment, which was owing to the breakage of ortho ester bonds, and

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 51503001), the Research Foundation for Key Program of Education Department of Anhui Province of China (No. KJ2016A030), the Doctor Research Foundation of Anhui University (No. J10113190075), and the Academic and Technology Introduction Project of Anhui University (AU02303203).

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