Tumor microenvironment-responsive micelles for pinpointed intracellular release of doxorubicin and enhanced anti-cancer efficiency
Graphical abstract
Introduction
Over the past decade, nanocarriers have been extensively explored for the controlled and targeted delivery of hydrophobic chemotherapy drugs owing to their abilities in improving poor solubility of chemotherapy drugs, prolonging the half-life time of payloads in vivo and passively accumulating in the tumor regions via the enhanced permeability and retention (EPR) effect (Brannon-Peppas and Blanchette, 2004, Wang et al., 2012, Maeda et al., 2013). However, issues still remain because of the limitation of pinpointed drug delivery or on-demand drug release. For instance, it is highly desired to explore more effective smart nanocarrier to maximize intracellular delivery of anticancer drugs for superior cancer therapy owing to their complex intracellular trafficking to target sites. Consequently, it is a major challenge to develop a new smart nanocarrier with multiple stimuli-responsive features for optimal anticancer efficacy (Mura et al., 2013). To date, by taking advantage of the stimuli (pH, GSH and enzyme) of tumor microenvironment, various stimuli-responsive nanoparticles (NPs) have been investigated for targeted and controlled drug delivery. Among them, pH- or reduction- responsive NPs have been researched most frequently and comprehensively, owing to significant difference in pH and GSH between extracellular and intracellular conditions (Liu et al., 2014, Deng et al., 2015a). However, the majority of pH- or reduction- responsive NPs remain limited to exhibit complete and significantly rapid drug release within tumor cells. Few types of NPs have exhibited marked or visible changes in response to low pH or GSH, which is the foundation of rapid intracellular drug release.
Compared with normal tissues, tumor regions possess specific intrinsic biological signals, such as low pH and active redox/reduction atmospheres, which play important roles in triggering stimuli-responsiveness for rapid drug release (Yu et al., 2014). Among these signals, the pH stimulus of tumor regions has been widely studied. Most tumor tissues present a mildly acidic extracellular pH value (≈6.0) as a result of rapid anaerobic respiration and they can also yield more significantly acidic endosome/lysosome pH value (5.0–5.5), which is much lower than the pH values of normal tissues and blood circulation (Hu et al., 2013, Meng et al., 2014, Hou et al., 2015). Responding to the specific acidic micro-environment of tumor regions, pH responsiveness of NPs contributes to the extracellular charge reversal of nanocarriers or the intracellular rapid release of payloads and thereby improving the cellular uptakes or enhancing the cytotoxicity of chemotherapy drugs (Du et al., 2011). Many previous studies have reported that pH responsiveness could be easily triggered because of the protonation of amino and imidazole groups; or the cleavage of acid labile ortho ester, hydrazone, cis-aconityl, and acetal bonds within the acid microenvironment in tumor regions (Tong et al., 2014). Among various promising pH-responsive polymers, polymeric nanoscales based on histidine have emerged as a viable platform because the imidazole group in histidine yields a pKa value of around 6.5 and can be protonated in acidic endosomes. Consequently, this type of polymer can quickly forfeit the hydrophilic/hydrophobic balance and then rapidly release the payloads in cells. Additionally, the protonation of histidine can help NPs escape from endosomes owing to the “proton sponge” effect (Jiang et al., 2012, Zeng et al., 2012). Park et al. reported novel pH-responsive NPs self-assembled by N-acetyl histidine-conjugated glycol chitosan polymer for intracytoplasmic delivery of drugs; these NPs responded to the acidic intracellular microenvironment and had an effect on escaping from endosomes and releasing the payloads into cytoplasm, thereby inhibiting cell growth (Park et al., 2006).
Different from the pH-responsiveness in response to the extracellular or endosomal pH environment, reduction-responsiveness can be triggered by the reducing environment in the cytoplasm, where the concentration of glutathione (GSH) is 100- fold to 1000- fold higher (approximately 2–10 mM) than that of extracellular fluids (approximately 2–20 μM) (Cheng et al., 2011). Reduction-responsive NPs assembled by polymers containing disulfide bonds can disassemble quickly and release the payloads under the stimuli of high-concentration GSH in the cytoplasm (Williford et al., 2014, Deng et al., 2015b). Given the structure containing a central disulfide bond, cysteamine and 3, 3′-dithiodipropionic acid have been widely adopted as a reduction-responsive cleavable bridge to contact a hydrophobic segment (e.g., poly(caprolactone), stearic acid) with a hydrophilic one (e.g., polyethylene glycol, hyaluronic acid) to obtain an amphiphilic polymer (Meng et al., 2009). Compared with the insensitive control, the reduction-responsive micelles exhibited enhanced cytotoxicity and much higher tumor-targeting capacity (Li et al., 2012). However, so far, few studies have been successfully conducted on the contact of a pH-responsive segment with a hydrophobic or hydrophilic segment via a reduction-responsive cleavage bridge to obtain a polymer with dual pH/reduction sensitivity.
It is promising that the combinational application of pH and reduction responsiveness can obviously take advantage of both stimuli environments, including endosomal pH and high-concentration GSH in the cytoplasm, leading to a more rapid and more complete release of payloads (Wu et al., 2013). Compared with single pH or reduction-responsive NPs, dually pH/reduction-responsive NPs performed more efficiently in the delivery of chemotherapy drugs (e.g., doxorubicin (DOX)) because these NPs could cause stimuli-triggered release and markedly improve the uptake, thereby enhancing therapeutic efficiency. (Chen et al., 2011) Chen et al. developed dually pH/reduction-responsive biodegradable micelles based on the poly(ethylene glycol)-SS-poly(2,4,6-trimethoxybenzylidene-pentaerythritol carbonate) (PEG-SS-PTMBPEC) copolymer for intracellular delivery of DOX. The dually responsive micelles exhibited a boosted drug release in response to both low pH in the endosomes and high GSH in the cytoplasm, leading to the delivery of DOX into the nuclei and a significantly enhanced anticancer efficiency compared with the single reduction-responsive controls. (Chen et al., 2013)
To this end, novel dually pH/reduction-responsive micelles consisting of CMCH-SS-NA polymer and DOX was successfully synthesized to generate precise spatiotemporal responsiveness of intracellular drug release with minimalized undesired release at surrounding normal tissues. Carboxymethyl chitosan (CMCH), a hydrophilic derivative of chitosan (CS) with low toxicity, low immunogenicity, and perfect biodegradability (Upadhyaya et al., 2014), was selected as a hydrophilic segment of the CS derivatives CMCH-SS-NA. Cystamine was chosen as the reduction-responsive bridge for the disulfide bonds in its structure responding to intracellular GSH stimuli, whereas N-acetyl-l-histidine (NAHis) was applied as the pH-responsive group for its imazole group responding to specific low pH in tumor. DOX, a frequently employed chemotherapy drug with significant toxicity to the heart and a short half-life time in vivo (Yokoi et al., 2015), was trapped into the dually pH/reduction-responsive micelles. The DOX-loaded CMCH-SS-NA micelles (DOX/CMCH-SS-NA) with mono-dispersive size distribution were expected to deliver DOX to tumor tissues effectively via the EPR effect. Partial DOX released from DOX/CMCH-SS-NA in acidic endosomal compartments after endocytosis because of the protonation of NAHis. Thorough release behavior of DOX into the cytosol occurred because of the cleavage of intervening disulfide bonds responding to high GSH concentration in the cytoplasm after DOX/CMCH-SS-NA escaped from the endosomes. Finally, the basic chemo-physical properties, targeting effect, and anticancer activity of dually pH/reduction-responsive DOX/CMCH-SS-NA micelles in vitro and in vivo were investigated with non-pH/reduction-responsive DOX-loaded CMCH-lauramine micelles (DOX/CMCH-LA) for comparison (Scheme 1).
Section snippets
Materials, cell lines and animals
Chitosan, with a molecular weight (MW) of 8–10 kDa and deacetylation degree (DD) of 93.1%, was obtained from Xingcheng Biochemical Co., Ltd. (Nantong, China). Carboxymethyl chloride was purchased from Ai Keda Chemical Technology Co., Ltd. (Chengdu, China). 1-(3-Dimethylaminopropyl)-3-ethyl carbon carbodiimide hydrochloride (EDC·HCl), N-hydroxysuccinimide (NHS), and GSH were purchased from Aladdin-Reagent Co., Ltd. (Shanghai, China). 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium
Synthesis and characterization of CMCH-SS-NA and CMCH-LA
As shown in Fig. 1A, cystamine, with a disulfide linkage in the structure, was used as a reduction-responsive connecting bridge to conjugate the hydrophilic CMCH and the pH-responsive NAHis through its terminal amine groups. Firstly, CMCH was synthesized to improve the solubility of CS in neutral solutions and adhibit carboxyl groups to the backbones of CS. Then, some of the carboxyl groups of CMCH were modified with cystamine via amine-reactive coupling, thus forming CMCH-SS. To prevent the
Conclusions
In this study, a novel CMCH-SS-NA CS derivative with sharp pH/reduction sensitivity was successfully synthesized, in which the hydrophilic segment CHCM and the pH-responsive hydrophobic segment NAHis were connected with each other via a cleavable reduction-responsive disulfide bond. The dually pH/reduction-responsive micelles based on the CS derivative were developed for the pinpointed intracellular delivery of DOX. Consequently, rapid release of DOX from DOX/CMCH-SS-NA micelles could be
Declaration of interest
This work was supported by the National Natural Science Foundation projects of China (Nos. 81571788 and 81273463), Jiangsu Science and Technology Support Plan (BE 2011670), and Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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These authors contributed equally to the paper.