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0Materials Today • Volume 00, Number 00• December 2015 RESEARCH
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Tumor microenvironment and intracellular signal-activated nanomaterials for anticancer drug delivery
Ran Mo1* and Zhen Gu2,3,4 *
1 State Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009, China
2 Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, NC 27695, USA
3Center for Nanotechnology in Drug Delivery and Division of Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
4 Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
Cancer-associated stimuli-responsive nanosystems have been increasingly considered for the delivery of anticancer drugs, which primarily target the tumor microenvironment and/or intracellular elements to enhance intratumoral accumulation and promote drug release at the target site. The signals facilitating drug delivery include tumor and endocytic acidities, hypoxia, enzyme overexpression, as well as high levels of intracellular glutathione, reactive oxygen species, and adenosine-5'-triphosphate. This article reviews the current techniques and ongoing developments in anticancer drug delivery using these signals. In particular, the focus is placed on design strategies and methods of formulating novel nanoscaled materials. The merits and drawbacks of recent strategies, as well as potential future developments, are discussed.
Introduction
Nanomaterial-based drug delivery systems (nano-DDSs) are highly effective in enhancing the therapeutic efficacies of anticancer drugs, as well as reducing their adverse toxicities [1,2]. Nanocarriers, such as liposomes, polymeric nanoparticles, and inorganic nanovehicles, ensure that their anticancer drug cargoes are accumulated sufficiently in tumors after systemic administration, including small-molecule chemotherapeutics and macromolecular proteins/nucleic acids [3-5]. Drug delivery exploits the unique anatomical and pathological characteristics of solid tumors with extensive vascular permeability and insufficient lymphatic drainage, which is known to lead to the enhanced permeability and retention (EPR) effect [6]. Although various nano-DDSs have been successfully applied in experimental and preclinical animal or human models, the majority are not without their limitations, such as inferior pharmacokinetics, premature drug release into the systemic circulation, unwanted and nontargeted accumulation in healthy tissues, poor tumor penetration capacity, and uncontrollable drug release at the target site. To resolve these limitations, nanoparticles have been prepared using stimuli-responsive materials to enhance their predesigned functions
*Corresponding authors:. Mo, R. (rmo@cpu.edu.cn), Gu, Z. (zgu@email.unc.edu)
in response to the tumor microenvironment and/or intracellular signals, such as deshielding of polyethylene glycol (PEG), conversion of the surface charge, exposure of the cell-penetrating peptide (CPP) or tumor-targeting ligand, and control of drug release in an on-demand manner (Fig. 1) [7-13]. These physiological signals include the presence of acidity, redox potential (glutathione (GSH)), specific enzymes, reactive oxygen species (ROS), hypoxia, and adenosine-5'-triphosphate (ATP). Thus, this move towards stimuli-responsive materials can overcome crucial challenges to conventional nano-DDSs, thus enhancing the therapeutic efficacies and reducing the side effects. This review outlines the emerging methodologies that use these signals in designing nano-DDSs. The advantages and drawbacks of recent strategies, as well as potential future developments, are discussed.
pH-triggered drug release and tumor targeting
The existing acidic pH of the extracellular and intracellular environment of tumors is considered an appropriate internal trigger for the controlled release of anticancer drugs in tumor tissues and/or within the tumor endocytotic vesicles such as endosomes and lysosomes. In comparison with the pH values in the blood and healthy tissues (pH 7.4), the extracellular pH values in the tumor
1369-7021/© 2015 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). http://dx.doi.org/10.1016/j.mattod.2015.11.025
| Please cite this article in press as: R. Mo, Z. Gu, Mater. Today (2015), http://dx.doi.org/10.1016/j.mattod.2015.11.025
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FIGURE 1
Schematic illustration of the role of tumor microenvironment and intracellular signals in tumor targeting and controlled drug release after intravenous administration. The nanocarriers can be activated to enhance accumulation in tumor and cellular uptake in response to tumor extracellular signals, including low pH, overexpressing cancer-associated enzymes, and hypoxia. The anticancer drugs can be selectively released in response to intracellular signals, including low pH, redox potential, enzymes, ROS, hypoxia, and ATP.
FIGURE 2
have been found to range from 6.0 to 7.2 [14]. Furthermore, after endocytosis, rapid acidification is initiated via a proton influx. The intracellular pH in the subcellular organelles decreases to 5.0-6.0 in the endosomes and 4.0-5.0 in the lysosomes [15,16].
pH-sensitive DDSs have been developed by incorporating pH-labile chemical bonds into the polymer components or the polymer/carrier-drug conjugates, such as acetal [17-22], hydrazone [23-26], cis-acotinyl [27], orthoester [28,29], b-carboxylic acid amide [30], and glycerol ester groups [31,32]. These bonds are stable under neutral or alkaline conditions, but they tend to be hydrolyzed at acidic pH, thus enabling the release of the encapsulated drugs by disrupting the nanocarrier or the conjugated drugs through the degradation of the polymer/carrier-drug linkage. Kataoka et al. reported an intelligent small interfering RNA (siRNA)-polymer conjugate containing an endosomal acidic pH-cleavable maleic acid amide (MAA) for enhanced delivery of siRNAs [30]. This siRNA-polymer conjugate regenerated the poly-cation within the endosomes to escape, which was accompanied by the release of siRNA through the acid-responsive cleavage of MAA, which then sequence-specifically and significantly inhibited the growth of cancer cells. Recently, Gu and Mo et al. reported a novel cocoon-like DNA nanoclew that was integrated with an acid-sensitive nanocapsule containing DNase I for the controlled intracellular release of doxorubicin (DOX). This nanocapsule was synthesized by a long-chain single-stranded DNA containing numerous repeated GC-pair sequences for high DOX-loading ability (Fig. 2a) [32,33]. At the endo-lysosomal junction, DNase I was activated by the acid-responsive shedding of the polymeric shell, such that the nanoclew immediately self-degraded and rapidly released DOX for enhanced cytotoxicity against cancer cells.
Another approach involves the incorporation of protonatable groups including amino and carboxyl groups into the polymer to modulate the release of the encapsulated drug by inducing a
Acid-triggered drug release. (a) Cocoon-like DNA nanoclew integrated with an acid-sensitive nanocapsule encapsulating DNase I for controlled intracellular release of DOX. Reprinted with permission from Ref. [32] Copyright 2014 American Chemical Society. (b) pH-responsive reversible swelling-shrinking nanogel for deep penetration into tumors. Reprinted with permission from Ref. [36] Copyright 2014 Wiley-VCH.
structural change in the nanocarrier in response to the variation in pH. Once these groups become protonated below the acid dissociation constant (pKa), the nanocarrier is destabilized by the charge repulsion between the polymers [34-37] or the change in the am-photeric properties of the components [38-40]. To enable deep penetration into tumor, Mo and Zhang et al. recently developed a virus-like pH-responsive nanogel with a cross-linked polyelectrolyte core containing N-lysinal-N'-succinyl chitosan (NLSC) (Fig. 2b) [36]. The nanogel was capable of reversibly swelling/shrinking depending on the protonation degree of the amine and carboxylic acid in the zwitterionic NLSC at different pH values. When the strong protonation of the amine at acidic pH led to charge repulsion between the NLSC chains, the volumetric expansion facilitated the rapid release of the encapsulated DOX into the tumor cells, thus inducing cell death. After escaping from the dead cells, the contractive nanogel could repeatedly infect the neighboring tumor cells, thus permitting the drug to penetrate deep into the solid tumor.
Carriers have been ruptured by acid-triggered gas generation as a means of designing pH-responsive drug release systems [41-43]. Sung et al. used poly(D,L-lactic-co-glycolic acid) (PLGA)-based hollow spheres to encapsulate both DOX and sodium bicarbonate (NaHCO3) [41]. As a gas-forming agent, NaHCO3 reacted with the proton in the endosomes and lysosomes to rapidly produce carbon dioxide bubbles, causing the sphere shell to burst, followed by the rapid release of DOX. Multidrug resistance (MDR) in cancer was significantly overcome by the prompt release of DOX, which significantly enhanced the concentration of DOX inside the cancer cells [42].
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Inorganic nanosystems have been increasingly applied as promising nano-DDSs to potentially enhance the therapeutic efficacy of anticancer drugs. Mesoporous silica nanoparticle (MSN) is one of the most extensively investigated inorganic nanocarriers for use in stimuli-triggered DDSs. A variety of molecules, such as ring molecules [44-46], inorganic precipitates [47,48], polymers [49], proteins [50], and nanoparticles [51-53], have been used as 'caps' or 'gatekeepers' to seal the pores in MSN to prevent the release of drugs at neutral pH. However, these caps shed from the MSN when encountered with an acidic environment, through a change in their physical property or the degradation of cap-MSN linkers, thus enabling the controlled release of drugs. Chen et al. demonstrated the use of calcium phosphate (CaP)-capped MSNs for pH-responsive drug release, which could rapidly release the encapsulated DOX into acidic endosomal compartments by the acid-triggered degradation of the CaP composite on the surface of MSNs [48]. In addition, gold nanoparticles (AuNPs) and graphene oxide (GO) have also been applied as pH-responsive nano-DDSs for ensuring controlled drug release. For enhanced plasmonic imaging and intracellular drug release, Song et al. designed a plasmonic self-assembled vesicle using surface-enhanced Raman scattering (SERS)-encoded amphiphilic AuNPs that were grafted with hydro-philic PEG and hydrophobic PMMAVP of methyl methacrylate (MMA) and 4-vinylpyridine (4VP) [39]. The pyridine groups in 4VP were protonated at acidic pH below 5.4, which was the pKa value of PMMAVP, which underwent a hydrophobic-to-hydrophilic transition, in turn disrupting the vesicle and promptly releasing DOX. Feng et al. developed a dual pH-responsive GO-based nanocarrier loaded with DOX, which showed significantly increased cellular uptake at the tumor pH and enhanced DOX release at the lyso-somal pH, thus improving the cytotoxicity of DOX toward DOX-resistant cancer cells, compared with free DOX [54].
As described previously, the extracellular pH value in the tumor is relatively lower than that in the normal tissue, which forms the basis for 'smart' tumor-targeting strategies, such as acid-activated PEG deshielding, surface charge conversion, activation of nonspecific CPP, or exposure of specific ligands, for increased tumor cell uptake or efficient intracellular delivery. PEGylation is an effective approach to confer enhanced stability and prolonged circulation behavior to nanocarriers for the purpose of increased accumulation in tumors. However, these nanoparticles exhibit low cellular uptake due to the highly hydrophilic PEG corona with large steric hindrance. In addition, the flexible PEG chain can also affect intracellular delivery including endosomal escape or site-specific targeting. Acid-triggered deshielding of the PEG chain or layer has been increasingly considered to overcome this limitation, thus enhancing the intracellular accumulation and delivery of nano-particles. A variety of acid-labile moieties, such as hydrazone [55,56], ester [57], and imine bonds [58], have been covalently conjugated between the nanoparticle and the PEG to confer an acid-active dePEGylation capability to the nanoparticle, which is highly stable at neutral pH but readily degraded at acidic tumor and endocytic pH values. Zhang et al. prepared a cyclodextrin dimer-connected self-assembled micelle with the capacity for tumor-induced targeting based on the host-guest interaction [58]. The protecting conjugated PEG chain was removed and the shielded RGD tumor-targeting ligand was exposed at the tumor site by the acid-triggered degradation of the benzoic-imine linkage. With the thermo-triggered phase transition, the micelles underwent disruption and promptly released the loaded drugs. In addition to the covalent incorporation of acid-labile linkages, acid-triggered charge switches have been considered as an alternative dePEGylation strategy [59-61]. Yang et al. developed a PEG-shed-dable ternary nanoparticle for the tumor pH-targeted delivery of
FIGURE 3
pH-Responsive tumor targeting. (a) Ternary polyplex with tumor pH-responsive dePEGylation capacity for delivery of siRNAs. Reprinted with permission from Ref. [59] Copyright 2012 American Chemical Society. (b) Zwitterionic oligopeptide liposome responsive to extracellular and intracellular acidity of tumor for mitochondria-targeted drug delivery. Reprinted with permission from Ref. [68] Copyright 2012 Wiley-VCH.
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siRNAs (Fig. 3a) [59]. A cationic ssPEI800/siRNA complex was coated by a tumor pH-sensitive PEGylated anionic polymer (PPC-DA) using electronic adsorption. At a mildly acidic extracellular pH of the tumor, the amide bonds that were stable at neutral pH underwent rapid hydrolysis to expose the cationic amino groups of the PEGylated PCC, leading to the deshielding of PCC from the highly cationic ssPEI800/siRNA complex by electronic repulsion. This facilitated the cellular uptake of siRNAs and enhanced the antitumor activity.
Charge conversion is also an effective method of tumor-targeted delivery, wherein the surface charge reverses from negative for enhanced circulation to positive for increased cellular uptake or intracellular delivery in response to the environmental acidity. For drug delivery responsive to extracellular or intracellular tumor acidity, both Kataoka et al. [62-64] and Wang et al. [65-67] used a variety of smart nanocarriers with a negative surface charge at neutral pH but positive charge at the tumor or endosomal pH. Mo et al. developed a mitochondrial-targeted liposome (HHG2C18-L) that responded sequentially to the tumor microenvironmental and intracellular compartmental pH;this liposome contained a synthetic functional lipid, 1,5-dioctadecyl-L-glutamyl 2-histidyl-hexahydrobenzoicacid (HHG2C18) (Fig. 3b) [68]. The negative surface charge of HHG2C18-L at physiological pH reversed to positive at the tumor pH to enhance the tumor cell uptake. Following endocytosis into the endosome, the histidine group exerted a proton-buffering effect for endosomal escape, whereas the carboxylic acid group was detached by the degradation of hexahydro benzoic amide. After escaping into the cytoplasm, the liposome, which possessed a higher positive charge as the carbox-ylic acid group was removed, preferentially accumulated on the mitochondria driven by electrostatic adsorption. Temsirolimus-loaded HHG2C18-L exhibited a considerably higher anticancer efficacy than a temsirolimus-loaded control liposome unresponsive to pH. Furthermore, incorporating a synthetic PEGylated lipid ensure the persistence of the liposome in the blood, in turn increasing the inhibitory effect on tumor growth [69].
Tumor-targeted drug delivery can also be achieved with tumor acidity-responsive deactivation to activation of the CPPs with non-tumor-specific penetration capacities. Shen et al. developed an acid-activated TAT-modified PEG-b-PCL micelle, in which the amines of lysine residues in TAT were amidized to succinyl amides for deactivating its function [70]. The acidity in the tumor stroma or endocytic vesicles promoted the hydrolysis of the succinyl amides and the activation of TAT to enhance cell penetration and nuclear targeting.
GSH-triggered drug release
GSH, a g-glutamyl-cysteinyl-glycine tripeptide, is the most abundant thiol in mammalian cells with an important role in the main biological functions [71]. The concentration of GSH is as high as 0.5-10 mM inside cells in the presence a highly reducing intracel-lular milieu. In sharp contrast, GSH is present at extremely low concentrations of approximately 2-20 mM in the blood or extracellular matrices. Such a significant gradient in the reduction potential from the extracellular environment to the intracellular cytosol can be harnessed to program the intracellular release of molecules [72]. More importantly, compared with healthy tissues, the tumor tissue has been found to be highly reducing and
hypoxic, with the intracellular GSH concentration being at least fourfold higher than that in the normal cells [73]. This significant difference in the concentration of GSH between the normal and tumor cells is relevant for tumor-specific drug delivery [74].
Redox-responsive nanocarriers have been used to confer sensitivity to the intracellular reducing potential for the burst release of encapsulated or conjugated drugs into tumor cells, such as liposomes [75,76], polymersomes [77], polymeric nanogels [78,79], micelles [80-87], dendrimers [88,89], protein nanocapsules [9092], MSNs [93-95], and AuNPs [96-98]. The materials used to fabricate nanocarriers contain characteristic disulfide (S-S) bonds in the backbone, side chain, cross-linker, or polymer-drug linkage for intra-/intermolecular conjugation. While the disulfide bond is highly stable in extracellular environments with low levels of GSH, it tends to be rapidly cleaved in highly reducing intracellular environments via a GSH-mediated thiol-disulfide exchange reaction, which may cause the disassembly of nanocarrier, thus releasing the cargoes.
Thayumanavan et al. synthesized a self-cross-linked polymeric nanogel composed of pyridyl disulfide (PDS)-grafted oligoethyle-neglycol (OEG)-based polymers by intra-/intermolecular disulfide cross-linking [78], which was highly responsive to the intracellular reducing environment, thus ensuring efficient release of DOX to achieve cytotoxicity against cancer cells. Recently, Tang et al. developed a tumor-targeted and redox-responsive protein nano-capsule for the intracellular delivery of recombinant p53 protein (Fig. 4a) [92]. The nanocapsule covalently conjugated with the tumor-targeting ligand, a luteinizing hormone-releasing hormone peptide, could specifically accumulate in tumor cells with overexpression of the corresponding receptors and efficiently release the p53 protein in response to the high intracellular levels of GSH that induce p53-mediated apoptosis.
Feng et al. used an intermolecular disulfide-cross-linked polymeric network as a gatekeeper to control the release of the encapsulated cargoes from MSN in response to redox signals [93]. Poly(N-acryloxysuccinimide) polymers were grafted onto the surface of MSN, and cystamine was used to cross-link the polymer chains for forming a polymeric network to block the MSN openings. In a reducing environment, the disulfide linker in cystamine was cleaved and the pore entrance was reopened, thus leading to a redox-responsive release of cargo.
In addition to the disulfide linkers, more reactive diselenide (Se-Se) bonds have been integrated into nanocarriers to attain redox responsiveness [100-102]. Xu and Zhang et al. synthesized a redox-sensitive micelle that was self-assembled by an amphiphilic diselenide-containing triblock copolymer (PEG-PUSeSe-PEG) [100], which collapsed to release molecules in a reduction/oxidation-activated manner. However, the limitation of diselenide-based redox-responsive nanocarriers is the difficulty of incorporating diselenide bonds into polymers.
Furthermore, using a novel strategy independent of the thiol-disulfide switch, Pei et al. developed a redox-responsive cationic vesicle consisting of ferrocenium-capped amphiphilic pillar[5]ar-ene for the co-delivery of DOX and siRNA (Fig. 4b) [99]. As the GSH-responsive structural element, the ferrocenium cation could be reduced to ferrocene by GSH, resulting in the disassembly of the nanocarrier through an amphipathy-to-hydrophobicity conversion and the consequent release of DOX and siRNA.
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Reduction-triggered drug release. (a) Tumor-targeted and redox-responsive protein nanocapsule for intracellular delivery of recombinant p53 protein. Adapted with permission from Ref. [92] Copyright 2014 American Chemical Society. (b) Redox-responsive cationic vesicle composed of ferrocenium-capped pillar[5]arene for co-delivery of DOX and siRNA. Adapted with permission from Ref. [99] Copyright 2014 Wiley-VCH.
Enzyme-triggered drug release and tumor targeting
Enzymes play significant roles as macromolecular biological catalysts in the majority of metabolic processes. The pathology of many diseases including cancer is associated with the malfunction of enzymes or the dysregulation of their expression. For the purpose of on-demand drug release, enzyme-responsive nanosys-tems for anticancer drug delivery have been developed by incorporating specific moieties that can be recognized and degraded by the enzymes overexpressed in the extracellular or intracellular environment of the tumor, compared with the enzyme levels in normal tissues [103-106]. The most commonly studied cancer-associated enzymes acting as favorable triggers include matrix metalloproteinases (MMPs) [107], hyaluronidase (HAase) [108110], and cathepsin B [111-113]. Zhao and Wang et al. used gelatin, a biocompatible substance that can be degraded by MMPs, as a gatekeeper conjugated on the surface of DOX-loaded MSNs (DOX/MSNs) for tumor-site-specific drug release [107]. The release of DOX from MSNs was significantly enhanced in the presence of MMP-9, which was efficiently delivered into the MMP-overexpres-sing colon carcinoma (HT-29) cells, produced greater cytotoxicity against HT-29 cells rather than the normal liver (LO2) cells. Chen et al. developed polyvalent nucleic acid-capped MSNs for controlled intracellular drug release, which efficiently released the encapsulated cargoes in response to either intracellular DNase I or thermal stimuli [114]. Recently, Gu et al. developed a core-shell gel-liposome complex (Gelipo) for sequential and site-specific delivery of tumor necrosis factor-related apoptosis-inducing li-gand (TRAIL) and small-molecule DOX [109]. Gelipo was composed of a liposome core for loading DOX and a cross-linked HA
shell for encapsulating TRAIL, which could sequentially release TRAIL extracellularly by the HAase-mediated degradation of the HA shell and release DOX intracellularly in response to the endosomal acidity. Gelipo in combination with two agents showed a significantly greater inhibitory effect on inhibiting the MDA-MB-231 tumor growth, compared with the DOX solution and Gelipo loaded with DOX alone. Mao and Yang et al. used cathepsin B, a lysosomal cysteine protease that is highly expressed in various cancer cells, as an endogenous trigger to induce intracellular drug release [112]. A urokinase plasminogen activator receptor-targeted magnetic iron oxide nanoparticle (ATP-IONP-GEM) that was conjugated with gemcitabine (GEM) via a cathepsin B-degradable tetrapeptide linker (GFLG) was proposed for targeted cancer therapy and magnetic resonance imaging (MRI). This theranostic nanocarrier facilitated an enzyme-responsive release of GEM intracellularly and a contrast-enhanced MRI of tumors. When administered intravenously, ATP-IONP-GEM significantly inhibited the growth of pancreatic tumor in xenograft mice.
In addition, the significant difference in protease expression intracellularly and extracellularly can provide a base for the design of enzyme-stimulated drug release systems [115-118]. Bernardos et al. prepared a saccharide-capped MSN as an enzyme-responsive nanosystem for controlled drug release intracellularly [115]. The grafted starch derivatives and lactose were conjugated on the surface of MSNs as gatekeepers, serving as the substrates of an endoprotease b-D-galactosidase. The DOX-loaded MSNs were highly cytotoxic against human cervical cancer (HeLa) cells, which was attributed to the enzyme-triggered degradation of saccharide and the subsequent release of DOX. Recently, Mo and Gu et al. developed a
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graphene-shuttled co-delivery system of TRAIL and DOX for a combination treatment of cancer (Fig. 5a) [118]. TRAIL was cova-lently conjugated with a PEGylated GO nanosheet via a furin-cleavable peptide to reduce the incidence of premature release, with the GO nanosheet displaying a high DOX loading capacity. The GO nanocarrier released TRAIL extracellularly by the furin-mediated hydrolysis of the transmembrane peptide, liberating DOX intracel-lularly by increasing the endocytic pH in a site-specific manner. The co-loaded GO containing furin-degradable peptide showed greater antitumor activity than that with the nondegradable PEG linker.
In addition, the overexpressed enzymes in the extracellular matrix of tumors have also been used as triggers in the deshielding of PEG, exposure of the ligand, or activation of CPP to enhance the tumor-targeting effect of nanosystems [119-126]. Torchilin et al. developed a multifunctional liposome containing TAT-PEG2000-PE and antibody-conjugated PEG3400-PE (mAb2C5-PEG3400-PE) with an MMP-cleavable peptide linker between PEG3400 and PE [119]. The long PEG3400 chain inhibited the cell-penetrating activity of TAT, which was conjugated on the short PEG2000
FIGURE 5
Enzyme-responsive drug release and tumor targeting. (a) Graphene-based nanocarrier for furin-responsive extracellular release of TRAIL. Reprinted with permission from Ref. [118] Copyright 2015 Wiley-VCH. (b) A tumor microenvironment-responsive nanoparticle functionalized by a hairpin-type pH and MMP dual-activatable CPP (dtACPP) for a combination treatment if chemotherapy and gene therapy. Reprinted with permission from Ref. [126] Copyright 2013 American Chemical Society.
chain in the blood circulation. However, once in the MMP-rich tumor matrix, the PEG3400 chain was removed by the MMP-specific cleavage of peptides, thereby exposing the TAT on the liposomal surface for effective cellular internalization. For a combination treatment comprising chemotherapy and gene therapy, Jiang et al. proposed a tumor microenvironment-responsive nano-particle functionalized by a hairpin-type pH and MMP dual-acti-vatable CPP (dtACPP) (Fig. 5b) [126]. In dtACPP, the cell-penetrating activity of CPP was restricted by a pH-sensitive masking peptide that was covalently conjugated to CPP via an MMP-hydrolyzable peptide. The masking peptide displayed a negative charge at neutral pH to efficiently shield CPP via electronic interaction, although the peptide was neutralized at the tumor pH to eliminate the shielding effect. After the MMP-driven cleavage of the linker, dtACPP was activated to CPP for increased intracellular accumulation.
ROS-triggered drug release
Studies have shown that cancer cells, compared with normal cells, constantly produce high levels of ROS derived from by-products of aerobic metabolism due to oncogenic transformation, such as hydrogen peroxide, hydroxyl radicals, and superoxide [127,128]. The sensitivity of ROS-responsive drug carriers to these ROS can be enhanced for site-specific drug release, which mainly contain characteristic groups such as boronic ester [129-132], thioketal [133-135], sulfide [136-138], selenium [139-142], and ferrocene groups [143-146]. Almutairi et al. developed a self-degradable nanoparticle composed of a synthetic polymer with a boronic ester used as an ROS-responsive unit for controlled release of cargo (Fig. 6a). At the inflamed or cancerous tissue, the boronic ester cap was removed to produce phenols, which consequently led to a quinone methide rearrangement, thereby disrupting the nanocarrier and releasing the cargo. The nanopar-ticle was highly sensitive, undergoing complete degradation into small molecules under conditions of oxidation; however, the safety of these degradation products for clinical use must be evaluated further. Xia et al. synthesized a cationic polymer (PATK) with ROS-cleavable thioketal linkages for mediating intracellular gene delivery (Fig. 6b) [134]. The DNA/PATK polyplex formed after endocytosis was dissociated by the cleavage of thioketal bonds in PATK with the accompanying release of the complexed DNA, thus leading to effective gene transfection in the prostate (PC3) cancer cells. Huang and Yan et al. developed an ROS-sensitive nanocarrier with a selenide-containing hyperbranched polymer (Fig. 6c) [142]. The DOX-loaded nanovehicle was rapidly disrupted in the target cancer cells under intracellular conditions of oxidation, thus promptly releasing DOX for inducing cell apoptosis.
Hypoxia-triggered drug release
The tumor microenvironment is typically hypoxic due to an imbalance between the oxygen supply and consumption, compared with normal tissues [147]. Tumor hypoxia plays an important role in enhancing MDR in cancer as well as malignant invasiveness and metastasis, leading to progression of cancer and poor prognosis. Due to its unique function, hypoxia is a promising target for cancer imaging and therapy [148-151], although hypoxia-responsive DDSs have not been investigated extensively thus far. Park et al. recently developed a self-assembled
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FIGURE 6
ROS-triggered drug release. (a) Self-degradable nanoparticle for controlled drug release in response to hydrogen peroxide. Reprinted with permission from Ref. [130] Copyright 2012 American Chemical Society. (b) Cationic polyplex containing thioketal linkages for ROS-activated intracellular DNA release. Reprinted with permission from Ref. [134] Copyright 2013 Wiley-VCH. (c) ROS-responsive nanocarrier composed of selenide-containing hyperbranched polymer for on-demand intracellular DOX release. Reprinted with permission from Ref. [142] Copyright 2013 American Chemical Society.
nanoparticle composed of carboxymethyl dextran integrated with a hydrophobic 2-nitroimidazole derivative for the purpose of hypoxia-triggered drug release [152]. The nanoparticle facilitated the sustained release of the encapsulated DOX under normoxic condition, but it significantly promoted drug release under hyp-oxic conditions, leading to a greater cytotoxic effect against hyp-oxic cells than normoxic cells. Systemic administration of the DOX-loaded nanoparticle displayed significant antitumor efficacy compared with the DOX solution. Wang et al. developed a nano-carrier composed of PEG, azobenzene, PEI, and DOPE units for the hypoxia-triggered dePEGylation of the nanocarrier and enhanced cellular uptake of siRNA, which exhibited hypoxia-activated gene-silencing capacity both in vitro and in vivo [153].
ATP-triggered drug release
ATP, also termed as the 'molecular unit of currency' of energy transfer in cells, is a molecule fundamental in cellular signaling and metabolism. The concentration of ATP is <5 mM in the extracellular fluid, whereas it is as high as 1-10 mM intracellularly [154156]. The significant gradient in the ATP level between the extracellular and intracellular environments has been recently applied in the design of ATP-triggered drug release systems.
Kataoka et al. designed a phenylboronate-modified polyion complex (PIC) micelle for the ATP-triggered intracellular release of siRNA (Fig. 7a) [157]. The siRNA was encapsulated in the PIC
micelle through the binding between its ribose and the phenyl-boronate. This micelle was remarkably stable due to the siRNA cross-linking at the extracellular level of ATP, but it was disrupted at the intracellular level of ATP via competitive binding between the ribose of ATP and phenylboronate. Biswas et al. developed a protein-based nanotube for ATP-responsive drug release [158]. This nanotube was prepared by coordinately linking many barrel-shaped chaperonins with magnesium ions (Mg2+), which prevented the biological degradation of cargo molecules in the tubular structure. After entry into the cells, the nanotube disassembled due to the conformational variation in the chaperonin units, which was caused by the hydrolysis of ATP into adenosine-5'-diphosphate (ADP), hence leading to the selective release of guests in the cells.
ATP aptamers have been extensively used to detect ATP with a variety of fluorescent, electrochemical, and colorimetric sensors [159-161]. For the purpose of selective drug release in response to the intracellular level of ATP, Gu et al. designed an ATP-respon-sive nanogel containing a complex core of an ATP aptamer-incorporated DNA scaffold with protamine and a cross-linked HA shell based on the interaction between the ATP aptamer and the target ATP (Fig. 7b [162]. This nanogel accelerated the release of the intercalating DOX from the DNA duplex via a structural switch from the duplex to the aptamer/ATP complex under an ATP-rich environment, thus showing a significantly
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ATP-triggered drug release. (a) Polyion complex micelle functionalized with phenylboronate for ATP-triggered siRNA release. Reprinted with permission from Ref. [157] Copyright 2012 Wiley-VCH. (b) Polymeric nanogel functionalized with an ATP aptamer-incorporated DNA scaffold for ATP-triggered anticancer drug delivery. Reprinted with permission from Ref. [162] Copyright 2014 Nature Publishing Group.
higher cytotoxicity than the non-ATP-responsive nanocarrier. When functionalized with an HA shell acting a tumor-targeting ligand, the nanogel was capable of enhancing the inhibitory effect on the tumor growth in MDA-MB-231 tumor-bearing mice. With the aim of promoting ATP-driven drug release, Gu et al. subsequently developed a liposomal co-delivery system consisting of a fusogenic liposome with a DOX-intercalated ATP apta-mer-incorporated DNA motif and a liposome encapsulating ATP by delivering exogenous ATP [163]. The direct delivery of the liposomal ATP facilitated the release of DOX from the fusogenic
liposome in the acidic endocytic vesicles by an acid-induced membrane fusion, thus increasing the therapeutic efficacy both in vitro and in vivo. Recently, Mo et al. designed a DNA-GO hybrid nanoaggregate cross-linked by an ATP aptamer, which also exhibited intracellular ATP-triggered DOX release [164]. The ATP apta-mer can also be used as a gatekeeper to prevent the premature release of cargoes from MSNs, as the binding of ATP to the ATP aptamer may lead to the removal of the gatekeepers, thus triggering the cargo release in response to the surrounding ATP [165-167].
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Summary and outlook
The recent advancements in nanotechnology, materials science, and chemistry drive the design and application of stimuli-triggered nanodevices for anticancer drug delivery, which facilitates the delivery of therapeutic agents in a spatiotemporal and on-demand manner for the treatment of cancer. The differences in the physiological and biological characteristics between the tumor and healthy tissue are being increasingly studied as the basis for nanosystems sensitive to unique endogenous signals from the extracellular and intracellular environments of tumors for enhanced tumor-specific targeting and controlled drug release.
The exploitation of the tumor microenvironment and/or the integration of cellular signal-activated nano-DDSs with elaborately designed synthetic materials have proved a promising alternative for the targeted delivery of anticancer drugs, wherein drugs are released in response to these triggers at the appropriate time and place. Despite the current remarkable advances, only a minority of these nanovehicles have been examined in preclinical animal models in vivo, and few have been applied clinically. For instance, nano-DDSs such as thermosensitive liposomes (ThermoDox) and iron oxide (NanoThermo) are currently under clinical trials, both of which respond to the exogenous rather than endogenous stimuli [8]; ThermoDox and NanoThermo are currently being investigated for the treatment of breast and liver cancer in phase III clinical trials, respectively. This is mainly because of the lack of sufficient evidence and in-depth studies on the safety, biocompat-ibility, and degradability of these novel materials, which considerably hinders their application from animal to human models. Biocompatible or biodegradable materials that can selectively respond to physiological signals can help reduce the adverse and toxic effects of the nanocarriers. A comprehensive evaluation of the safety of the materials and nanocarriers must be performed before being put into clinical practice, which includes aspects such as pharmacokinetics, biodistribution, metabolism, excretion, and long-term toxicity. Moreover, the production of these 'smart' nanocarriers on an industrial scale is greatly limited by the complexity of structural design and the laborious process of synthesizing materials. In addition, a variety of physiological signals have already been used for controlled drug release. However, endogenous signals including pH or GSH in the extracellular or intracel-lular environments of tumors are difficult to control, varying in different individuals. For clinical practice, the most effective signal should be considered to attain the ideal efficacy [168,169].
Acknowledgments
This work was supported by the Natural Science Foundation of Jiangsu Province of China for Distinguished Young Scholars (BK20150029), the Six Talent Peaks Project of Jiangsu Province of China, the Jiangsu Specially-Appointed Professors Program, the CPU High-Level Talent Program, the start-up package from CPU to R.M., NC TraCS, NIH's Clinical and Translational Science Awards (CTSA, 1UL1TR001111) at UNC-CH, and the start-up package from the Joint BME Department of UNC-CH and NC State to Z.G.
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