Review For reprint orders, please contact [email protected]

pH-sensitive drug-delivery systems for tumor targeting Drug-delivery system responses to stimuli have been well investigated recently. As pH decrease is observed in most solid tumors, drug-delivery systems responsive to the slightly acidic extracellular pH environment of solid tumors have been developed as a general strategy for tumor targeting. Drug vehicles that are sensitive to acidic endosome/lysosome pH have been constructed for efficient drug release in tumor cells. This review explains the mechanisms of acidic pH in the tumor microenvironment and endocytic-related organelles, endosomes and lysosomes. Nanoparticle responses to acidic extracellular pH are discussed, along with approaches for improving tumor-specific therapy. Endosome/lysosome pH-triggered vehicles are reviewed, which achieve rapid drug release in tumor cells and overcome multidrug resistance. Cancer is one of the major global public health problems, and is the second leading cause of death following heart diseases. A total of 1,638,910 new cancer cases and 577,190 deaths from cancer were projected to occur in the USA in 2012 by the American Cancer Society [1]. Besides surgical resection and radiation therapy, chemotherapy plays an important role in treating malignant neoplasm. However, one of the long-standing problems in chemotherapy is the lack of tumor-specific targeting, which may result in severe systemic toxicity, causing a decline of immune function, liver damage, myelosuppression, inflammatory reaction and so on. As cancer cells proliferate rapidly, the quick formation of new blood vessels results in abnormal pathological states such as pericyte deficiency and aberrant basement membrane formation, leading to enhanced vascular permeability [2]. Moreover, lymphatic drainage in cancer is absent, which contributes to particle retention at the cancerous site. This phenomenon, which is called the enhanced permeability and retention (EPR) effect has been utilized in passive tumor targeting [3,4]. Drug-delivery systems with a size range of 20–200 nm can be accumulated inside the tumor interstitial space via the EPR effect. To improve the passive tumor targeting efficiency, drug-delivery systems have been modified with materials such as PEG, mediating a long circulation of nanoparticles (NPs) and contributing to a better biodistribution of anticancer drugs. However, passive targeting alone cannot resolve the problem of cancerous tissue selectivity. Combining passive targeting with active

targeting brought a new strategy for tumor therapy. Active targeting is a strategy based on the overexpressed tumor-specific receptors such as the EGF receptor [5,6], the ErbB2 receptor [7], the leptin receptor [8] and the transferrin receptor [9,10]. Folic acid (FA) [11,12], certain antibodies (monoclonal antibodies) [13], artificial peptides T7 [9,10] and many other ligands that specifically bind to tumor overexpressed receptors could be added onto drug-delivery systems, contributing to an improved tumor-targeting effect. As there has not been a receptor that is expressed in tumor cells exclusively, their expression in normal tissue may result in low drug-delivery efficiency and severe toxicity. In order to address this challenge, novel drug-delivery systems capable of releasing their payload in response to stimuli have received much attention in recent years. Tumor hypoxia and pathological pH change in tumors are the stimuli that have been well investigated. Metal-based prodrugs with redox activation, nanomaterials with enzyme-response and light-triggered nanocarriers have been reviewed elsewhere [14–16]. This review mainly focuses on pH-sensitive tumor-targeting drug-delivery systems, which respond to the tumor extracellular acidic pH tissues and intracellular organelles such as endosomes and lysosomes. The mechanisms of physiological acidic pH of intracellular compartments and pathological low extracellular pH (pHe) in cancerous tissues will be elucidated. Strategies for extracellular and intracellular pH-triggered tumor targeting will be reviewed by providing several mechanisms and examples illustrating their application.

10.4155/TDE.13.120 © 2013 Future Science Ltd

Ther. Deliv. (2013) 4(12), 1499–1510

Xi He, Jianfeng Li, Sai An & Chen Jiang* Key Laboratory of Smart Drug Delivery, Ministry of Education, Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai 201203, China *Author for correspondence: Tel.: +86 2151 980 079 Fax: +86 2151 980 079 E-mail: [email protected]

ISSN 2041-5990

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production and poor proton clearance, the tumor extracellular pH shows a slight decrease compared with physiological pH, which could be utilized in improving tumor-targeting specificity.

pH environment in tumor „„Low extracellular pH in cancerous tissues & the mechanism Compared with normal tissue, tumor cells can take up 12-fold glucose and have increased aerobic glycolysis, but use a smaller fraction of glucose for mitochondria oxidative phosphorylation even, when oxygen levels are normal, thus producing more lactate and less ATP. This phenomenon was first observed by Warburg in the 1930s, and is called the Warburg effect [17]. Following glycolysis, lactic acid is exported from cells via the H+-monocarboxylate co-transporter, mediating an increased amount of protons in the tumor microenvironment [18]. Besides lactic acid, large amounts of CO2, which is produced by tumor cells via oxidative metabolism, is another major source of tumor extracellular acidity [19]. However, attributed to poor lymphatic drainage and elevated interstitial pressure of tumors, metabolic acids (lactate and CO2) exported from the cancer cells into the interstitial fluid cannot be exported to the blood rapidly [20]. Resulting from the increased proton production and poor proton clearance, pHe illustrated a low value range of approximately 6–7 [21]. „„Acidic

endosome/lysosome pH in cancer cells Other than mild acidic extracellular pH in tumor tissue, an even greater pH decrease can be found in intracellular compartments compared with normal tissue (pH 7.4). The most widely known acidic organelles are lysosomes and endosomes, which play an important role in endocytic pathways. Many groups have reported that the lysosome and endosome environment are maintained at a pH of 4–5 and 5–6, respectively, while the first direct evidence indicating acidity of endosomes was provided by Tycko and Maxfield. The mechanism of the internal acidic environment of endosomes and lysosomes is through the presence of H+-ATPases [22]. Several drugs and drug carriers are taken up by endocytosis and trapped in endosomes and lysosomes. Developing drug-delivery systems responsive to such a pH decrease in intracellular compartments gives another resolution to improving the efficiency of tumor-targeting therapy. pHe triggered tumor targeting NPs Although modifying tumor-targeting ligands to drug-delivery systems could improve the poor cellular internalization of anticancer drugs to a certain extent, the expression of receptors in 1500

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both tumor cells and normal tissues would still cause toxicity and hamper the efficiency of cancer therapy. As pHe in the tumor site is lower than normal tissues, this phenomenon can be used for acid-triggered tumor targeting [18]. With this in mind, combining a pHe responsive ability with a tumor-targeting ability could improve the specificity of tumor therapy. The approaches to tumor targeting via acidic pHe include modifying pH-sensitive peptides [23–25], utilizing pH-triggered ionized materials [26–28], and exposing ligands by a shielding/deshielding mechanism [29] or by a pop-up mechanism [30]. „„pH-sensitive

peptides Inspired by recombinant technology, synthetic peptides, which have a pH-activated cell-penetration ability, have been constructed. GALA, pHLIP, histidine-containing acidic activated cell-penetrating peptides (CPPs) and other pHsensitive synthetic peptides have been widely used as ligands in tumor-specific targeting. GALA is a 30-amino acid, pH-sensitive fusogenic peptide, with a glutamic acid–alanine–leucine–alanine (EALA) repeat that also contains a histidine and tryptophan residue. The sequence of GALA is WEAALAEALAEALAEHLAEALAEALEALA A. The EALA repeat can present an amphipathic a-helix, which is essential in cell-penetrating behavior. At a neutral pH, the electrostatic repulsion between the carboxylic acid moieties of the glutamic acids (Glus) destabilize the helix, while at the acidic tumor environment the neutralization of Glus could promote helix formation, leading to the insertion of GALA into cell membranes [23,31]. Etzerodt et al. synthesized the GALA-derived lipopeptide DMDGALA by adding two myristoyl chains on the GALA [24]. Utilizing DMDGALA as a component in large unilamellar vesicles (LUVs), they successfully developed a pH-sensitive LUV, which clearly demonstrated fusion at pH 6.1, and a very low degree of fusion at pH 7.4. pHLIP is a water-soluble polypeptide derived from the bacteriorhodopsin C helix, which was found to insert across a membrane to form a stable transmembrane a-helix. The sequence of pHLIP peptide is AEQNPIY W A R Y A D W L F T T P L L L L D­L A L L V DADEGT. At physiological pH, the Asp residues are charged, enhancing peptide solubility and helping pHLIP anchor at the surface of membranes. However, at tumor acidic pHe, pHLIP can form a functional a-helix, inserting future science group

pH-sensitive drug-delivery systems for tumor targeting into the lipid bilayer and mediating cell penetration [25,32–34]. Vãvere constructed 64Cu-DOTApHLIP in positron emission tomography imaging of prostate tumors [32]. Han et al. constructed a pHLIP-mediated tumor targeting NP (dendrigraft l-lysines [DGL]-PEGpHLIP/pDNA) [29]. They conjugated pHLIP to DGL via bifunctional PEG, then condensed plasmid DNA (pDNA) to formulate pH-sensitive gene delivery NPs. The results of in vitro cellular uptake demonstrated the pH-sensitive behavior of pHLIP. When incubated at pH 7.4, DGL-PEG-pHLIP/pDNA NPs could not combine with hepatoma cells (Bel-7402). However, DGL-PEG-pHLIP/pDNA NPs illustrated significant combination with Bel-7402 cells at pH 6.0. In vivo distribution and gene transfection results also demonstrated enhanced tumor-targeting delivery efficiency. As histidine has a pKa of approximately 6.5, histidine-containing peptides have a net positive charge below pH 6.5, but are uncharged at pH 7.4. Therefore, a new kind of CPP containing histidines has been developed for response to the tumor extracellular acidic environment, achieving acid-activated cell-penetrating properties. Zhang et al. replaced all the lysines of transportan 10 with histidines, constructing TH (AGYLLGHINLHHLAHL[Aib]HHIL-NH2) [35]. At physiological pH, TH cannot bind to the cell membrane because of the loss of positive charge. While at the tumor acidic microenvironment, TH becomes protonated and cationic, achieving effective cell membrane binding by electrostatic attraction, resulting in more cellular uptake in Hela cells at pH 6.5 than that at pH 7.4. Other than GALA, pHLIP and histidinecontaining CPPs, Zaro et al. devised another pH-sensitive CPPs [36]. They utilized a highly pH-sensitive co-oligopeptide sequence fused to a model amphipathic peptide (MAP), which is a kind of CPP, to mask the cationic charge and to prevent the non-specific internalization of the CPPs in normal tissues (Figure 1). The pH-sensitive co-oligopeptide sequence consists of histidine–glutamic acid (HE) repeats. When exposed to the tumor acidic environment, the imidazole group in histidine will undergo a charge change from neutral to cationic, neutralizing the charge of HE peptides. Therefore, the cell-penetrating ability of MAP will be regenerated. In summary, this HE sequencemodified MAP fusion peptide could be utilized in several areas including tumor diagnosis, future science group

| Review

Tumor extracellular acidic environment pH decrease Masked conformation

Unmasked conformation Nanocomplex pH-sensitive sequence: (HE)10 CPPs: MAP

Figure 1. Mechanism of histidine–glutamic acid-model amphipathic peptide nanocomplex’s pH-sensitive behavior. At physiological pH, the nanocomplex will be in its masked form. At the tumor acidic environment, the histidine–glutamic acid sequence would be ionized, exposing CPPs for subsequent binding and internalization. CPP: Cell-penetrating peptides; HE: Histidine–glutamic acid; MAP: Model amphipathic peptide.

targeted macromolecular drug delivery and tumor-targeting gene-delivery systems. „„Shield/deshielding

mechanism Aiming to reduce nonspecific uptake and retain the tumor-targeting delivery efficiency, Han et al. designed DGL-PEG-T7-hydrazonediethylenetriamine pentaacetic acid (DTPA)/ pDNA NPs by a shielding/deshielding mechanism (Figure 2) [29]. DTPA, a hydrophilic and negatively charged molecule, which could prevent the contact of NPs with normal tissue cell membrane in neutral conditions, was chosen as the shielding molecule. Tumor-targeting peptide T7 was masked by DTPA via a pH-sensitive hydrazone linkage. Therefore, in neutral conditions, targeting ligand T7 is shielded to avoid nonspecific binding, while in the acidic tumor environment, the hydrazone bond (hyd) can be hydrolyzed to expose ligands for specific tumor

Tumor extracellular acidic environment pH decrease

DGL PEG Targeting ligand, T7 Shielding moiety, DTPA pH-sensitive bond, hydrazine bond

Figure 2. Mechanism of pH-sensitive shielding/deshielding behavior. At physiological pH, the nanoparticle will be shielded by diethylene triamine pentaacetic acid. At the tumor acidic environment, diethylene triamine pentaacetic acid would be deshielded to expose T7 for tumor targeting. DGL: Dendrigraft l-lysines; DTPA: Dendrigraft l-lysines-PEG-T7-hydrazonediethylenetriamine pentaacetic acid.

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Review | He, Li, An & Jiang Key Term pH-sensitive nanoparticle: Nanosized vehicle including, polymers or peptides that can respond to change in the pH of surrounding medium.

targeting. In the UV spectra, the absorbance region of DTPA for DGL-PEG-T7-hydrazoneDTPA disappeared after dialysis at pH 6.5, which indicates a successful deshielding behavior in the acidic tumor environment. In in vitro cellular-uptake experiments, DGL-PEG-T7-hydrazone-DTPA/pDNA NPs delivered very little pDNA into cells at pH 7.4, however, they did mediate more uptake at pH 6.5. Compared with nonshielding NPs, DGL-PEG-T7/pDNA NPs, which showed efficient gene expression in normal cells and tumor cells at normal conditions, DGL-PEG-T7-hydrazone-DTPA/pDNA NPs could reduce side effects on healthy cells and enhance the tumor-targeting delivery efficiency. Using hyd bonding and DTPA to achieve acidactive ligand-mediated tumor-targeting delivery strategies may be broadly applicable for other ligands as well. To demonstrate tumor cell selectivity via the erosion of layer-by-layer NPs, Poon et al. utilized a trilayer architecture of poly-l-lysine (PLL) modified with iminobiotin, followed by the linker protein neutravidin and biotin endfunctionalized PEG [37]. The neutravidin-biotin layer bridges PLL and PEG via a neutravidin– iminobiotin bond, which is stable at pH 8–12 but can be easily decomposed at pH 4–6. The PEG layer enables the layered NPs to avoid rapid reticuloendothelial system clearance and enhances their accumulation in tumor interstitials due to the EPR. When NPs accumulate in the acidic tumor environment, they gradually lose their PEG shells, allowing the exposed PLL layer to facilitate cellular uptake. Although PEG can be useful for systemic distribution, it may hinder subsequent cellular uptake in tumor cells, compromising efficiency of drug delivery to target cells. This

Tumor extracellular acidic environment pH decrease

PEG (3.4 kDa) PEG (2 kDa) polyHis (2 kDa) TAT peptide

Figure 3.Acid-induced pop-up targeting mechanism. At physiological pH, TAT is shielded by PEG. In the acidic tumor environment, poly-histidine is ionized, inducing TAT pop-up outside the PEG shell, mediating tumor targeting. polyHis: Polyhistidine.

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phenomenon is called the ‘PEG dilemma’ [38]. In order to overcome such a dilemma, Fella et al. [39] constructed a pH-sensitive NP via shielding/deshielding mechanism. They designed PEG-aldehyde-carboxypyridylhydrazone, N-hydroxysuccinimide esters to modify polyethylenimine (PEI) NPs. When NPs accumulate in acidic tumor environment, the hyd can be hydrolyzed, resulting in the removal of PEG and exposure of PEI NPs. The z potential of PEG aldehyde-carboxypyridylhydrazone, N-hydroxysuccinimide esters shielded NPS was approximately +4mV, while an increase to +33 mV at pH 5.0 was observed, which indicated the removal of the PEG shield and re-exposure of the positively charged PEI polyplexes. „„Ligand

exposure by pop-up mechanism Ligand exposure by pop-up mechanism is another strategy in designing a pH-sensitive drug delivery system. A pH-sensitive molecular chain actuator, a short poly(l-histidine) (polyHis) is widely used in this type of NP. Lee et al. produced TAT pop-up pH-sensitive micelles (PHSMpop-upTAT ), which is formed by self-assembly of a mixture of two block copolymers(poly(l-lactic acid)(3 kDa)-bPEG(2 kDa)-b-polyHis(2 kDa)-TAT and polyHis(5 kDa)-b-PEG(3.4 kDa) by dialysis methods [30]. The general schematic representation of acid-induced pop-up mechanism for PHSMpop-up TAT micelles is in Figure  3. At pH 7.4, TAT peptide can be anchored on the inside of the polymeric micelle via polyHis, thus being shielded by the PEG shell of the micelle. When PHSMpop-up TAT micelles accumulate in the tumor site, polyHis becomes charged and exposes TAT to the outside of PEG shell, facilitating specific cell penetration in tumor cells. At pH 7.4, PHSMpop-up TAT micelle uptake by human breast tumor MCF-7 cells was minimal. At pH 6.8, cellular uptake increased 70-fold compared with that at pH 7.4. At pH 7.4, the micelle demonstrated limited doxorubicin (DOX) release. However, at pH 6.4, PHSMpop-up TAT released approximately 78 wt% of the initial DOX. At the weakly acidic pH of 6.8, PHSMpop-up TAT showed eight-times higher cytotoxicity than that at physiological pH 7.4. Lee et al. also produced another pH-sensitive pop-up micelle system using biotin as targeting moiety. At physiological pH, biotin was shielded by PEG layers [40]. When the pH became slightly acidic, polyHis became charged and exposed future science group

pH-sensitive drug-delivery systems for tumor targeting the biotin, facilitating biotin receptor-mediated endocytosis in tumor cells. „„pHe

triggered tumor targeting via ionization mechanism Various ionizable monomers could be employed as materials for pH-sensitive micelles, including sulfonates [26], carboxylic acids [27] and amines [28]. 2-(disopropylamino) ethyl methacrylate (DPA) is an amine-containing monomer, which is highly biocompatible and pH sensitive with a pKa of 6.2 [41,42]. At physiological pH, DPA is hydrophobic due to its deionization, however, at pHe, it could be ionized and becomes hydrophilic. Peng et al. synthesized PEG methacrylate-co-DPA (PEGMA-co-DPA) by free radical polymerization [42]. The copolymer can selfassemble into NPs encapsulating hydrophobic photosensitizer, meso-tetra(hydroxyphenyl) chlorin (m-THPC). In vitro release of m-THPC at pH 6.0 was much faster than the neutral or basic environment at pH 7.0 or 8.0. The accumulated m-THPC release was 58, 45, 10, and 13% of initial concentrations at pH 5.0, 6.0, 7.0 and 8.0, respectively. This result showed the pH-sensitive property of PEGMA-co-DPA NPs. Amoozgar et al. utilized low molecular weight chitosan (LMWC) as pH-sensitive materials, covalently conjugating to poly(lactic-co-glycolic acid) (PLGA), synthesizing PLGA-LMWC polymers [43]. LMWC is a linear copolymer of glucosamine and N-acetylglucosamine. In the acidic environment of solid tumors, LMWC could be protonated and adhere to tumor cells via electrostatic interactions via glycocalyx on the cell membrane [44]. Encapsulating paclitaxel (PTX), PLGA-LMWC could self-assemble to form NPs with a size of 176.0 ± 45.2 nm. PLGALMWC NPs showed significantly stronger signals in ovarian cancer cells at pH 6.2 than at pH 7.4. In vitro cellular uptake results proved the pHe-targeting ability of PLGA-LMWC. Poly(b-amino ester) (PAE) is another polymer that could be ionized at mild low pH, which has been widely used in designing pH-sensitive degradable block copolymer [45–49]. Gao et al. synthesized the block copolymer PEG-PAEaqueous polymer isocyanate via Michael-type step polymerization method. During the reaction, the imidazole ring could be inserted into the copolymer, mediating pHe sensitivity [45]. At pH 7.4, the amino groups of PAE were fully deionized and became hydrophobic. Therefore, the pH-sensitive PAE moiety could be transformed into the hydrophobic core of the micelle, future science group

| Review

which would be shielded by hydrophilic PEG shells. When the pH decreased, PAE could be ionized and showed positive charge, resulting in more positive z potential of PEG-PAE-aqueous polymer isocyanate-derived micelle. Intracellular pH-triggered tumor-targeting NP To achieve efficient tumor therapy, anticancer drugs should not only be delivered to the tumor site and internalized into tumor cells, but they should also be released from lysosomes to the cytoplasm or nucleus. The pH value of early endosome and late lysosome is around pH 5–6 and pH 4–5, respectively, which is much more acidic than normal tissue [50]. A more advanced approach to efficient cytoplasmic drug release to cancer cells is to use drug vehicles that have lysosome pH-triggered drug release properties. Utilizing acid-cleavable chemical bonds and lysosome pH-sensitive materials to release drugs have been widely used approaches in intra­ cellular pH-triggered drug-delivery systems for tumor therapy. „„Directly

conjugating anticancer drugs via acid-cleavable linkages The hyd [51–55], cis-aconityl bond [56–59] and ether linkages [60,61] are acid hydrolyzable chemical bonds that have been widely used in constructing lysosomal pH-triggered drug-release NPs. DOX, an anthracycline antibiotic, which interacts with DNA by intercalation and inhibition of macromolecular biosynthesis, is commonly used in the treatment of a wide range of cancers [62,63]. The carbonyl group of DOX can react with hydrazine, formulating a pH-sensitive hyd. Many groups have successfully conjugated DOX to polymers via the hyd, achieving rapid release of the lysosomal triggered DOX [51,64–66]. Guo et al. developed a pH-sensitive tumortargeting micelle (FA-hyd micelles), using FA as target moiety and hyd conjugating DOX (Figure 4) [65]. Confocal laser scanning microscopy ana­lysis of FA-hyd micelles presented a stronger DOX fluorescence in the nucleus of KB cells compared with non-targeted micelles and acid uncleavable micelles. This demonstrates that FA-hyd micelles showed better efficiency of tumor chemotherapy. Besides the hyd, the cis-aconityl bond is another pH-sensitive linkage that has been widely used to conjugate anticancer drugs [57,67,68]. Du et al. developed a lysosomal pHtriggered prodrug denominated as FA-bovine www.future-science.com

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O FA

O

O

H N

O

CH2OH N

O

OH O O

HO

H OH

O

O

FA-PEG-b-PCL-hyd-DOX

CH3 OH NH2

Self assembly

OCH3

Endosome/lysosome environment pH decrease

Figure 4. pH-sensitive mechanism of folic acid-PEG-b-polycaprolactone-hydrazone bond-doxorubicin micelle. (A) Chemical structure of folic acid-PEG-b-polycaprolactonehydrazone bond polymer. Such amphiphilic polymer can self-assemble into nano-sized micelles. (B) After receptor-mediated endocytosis, the micelles encounter the acidic pH of the endosome/lysosome, mediating hydrolysis of hydrazone bond and release of doxorubicin. DOX: Doxorubicin; FA: Folic acid; hyd: Hydrazone bond; PCL: Polycaprolactone.

serum albumin (BSA)-cis-aconitic anhydridedoxorubicin [59]. BSA, which contains approximately 30–50 amino groups, was used as drug carrier. FA was chosen as targeting moiety for DOX delivery. DOX was conjugated to BSA via the cis-aconityl bond according to Shen’s method [69]. In order to investigate the pH sensitivity of FA-BSA-cis-aconitic anhydridedoxorubicin prodrug, the prodrug was incubated at different pH to monitor the release of free DOX. The amount of DOX released from the prodrug at pH 5.5 was approximately 4.5-fold higher than that at pH 7.2. This result suggested that the prodrug would be stable in blood circulation, whereas quickly release DOX after internalization into the acid lysosome. Other than the hyd and the cis-aconityl bond, ether linkages [60,61] also demonstrate the acidcleavable property. Das et al. developed an ultrasmall super-paramagnetic iron oxide nanocore conjugating the anticancer drug methotrexate (MTX) via ether linkage [61]. In in vitro drug release experiments, the overall release rate of MTX was much higher at a lower pH, whereas negligible drug release was observed at pH 7.4. Parrott et al. designed a novel silyl ether linkage that could conjugate hydroxyl group-containing drugs such as camptothecin, dasatinib and gemcitabine to NP carriers [60]. After exposure 1504

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to the acid the environment in lysosome, silyl ether linkage could be hydrolyzed to trigger the release of a high payload of drugs. „„Intracellular

drug release via acid unstable polymers Drug delivery systems based on titratable polymers can undergo a quick and controllable change in conformation upon a change in pH, mediating rapid endosome drug release. In most cases, the titratable polymers comprise alkyl chains to interact with the lipid bilayer, carboxylic acid groups for pH sensitivity and an amphiphilic character to achieve membranolytic properties [27]. There are two mechanisms of drug release in pH-sensitive vehicles. Encountering the pH decrease in endosomes and lysosomes, some polymers may be protonated, resulting in destabilization of liposomes and thereby promoting drug efflux to the endosome [70,71]. On the other hand, some polymers can fuse between the liposome and endosome/ lysosome membranes, promoting drug release to cytoplasm (Figure 5) [72–75]. Controlling drug release via polymer destabilization

pH-sensitive biodegradable polymers and pH triggered ionized polymers are two major types future science group

pH-sensitive drug-delivery systems for tumor targeting of materials widely used in pH-sensitive vehicles encapsulating anticancer drugs. Wu et al. developed core-crosslinked pH-sensitive degradable micelles using PEG-b-poly(mono-2,4,6-trimethoxy benzylidene-pentaerythritol carbonateco-acryloyl carbonate) (PEG-b-P[TMBPECco-AC]) diblock copolymer that contains acidlabile acetal [76]. At pH 5.0, the acetal groups in the TMBPEC backbone were hydrolyzed, resulting in fast intracellular drug release. Kim et al. developed a novel pH-sensitive polyacetal-based block copolymers for controlled drug delivery. Amphiphilic triblock copolymers were synthesized from a hydrophobic polyacetal, poly(ethyl glyoxylate) (PEtG) and hydrophilic methoxyl PEG (mPEG) [77]. The anticancer drug PTX was loaded in PEGPEtG-PEG triblock copolymer formulating pH-sensitive micelles. Encountering lysosomal acidic pH 5.0, the cumulative percentage of PTX release was 94.1%, which is much higher than that at pH 7.4. Therefore, PEtG ,which has acid hydrolyzable acetal linkages, has a potential use in designing pH-sensitive copolymers. Other acid degradable polymers containing acetal groups in backbones have been successfully developed [78]. Other than acetal groups, developing copolymers with hyds in backbones could also achieve destabilization and rapid drug release in the endosome or lysosome. Poly(e-caprolactone)-hydrazone-PEGhydrazone-poly(s-caprolactone), developed

| Review

by Zhou et al. [79], is a type of acid-degradable copolymer containing hyds. At acidic pH, the hyds would be hydrolyzed contributing to the destabilization of micelles and releasing drugs. Therefore, acid-labile polymers have received considerable attention in the field of endosome/lysosome-induced rapid drug release in cancer cells. The other types of polymers inducing vehicle destabilization are materials that could be ionized upon a decrease in pH value. Anionic polymers containing carboxylic acid groups, such as poly(acryilc acid) [80,81] and poly(methacrylic acid) [82,83] have been widely utilized. At physiological pH, they release protons and become hydrophilic. While at endosomal/lysosomal pH, they could be protonated, yielding a transition from hydrophilic to hydrophobic, resulting in destabilization of the derived vehicles. Poly(N,N´-dimethyl aminoethyl methacrylate), PAE, poly(4-vinylpyridine) and poly(histidine) [84–86], which contain tertiary amine groups or pyridine groups could be protonated as pH decreases, undergoing phase transition from hydrophobic to hydrophilic, inducing endosomal/lysosomal drug release. Kim et al. successfully constructed a pH-sensitive micelle utilizing poly(l-histidine)-b-PEG-folate or polyHis-b-PEG blended with poly(lactic acid) (PLLA)-b-PEG-folate [87,88]. Adjusting the amount of PLLA-b-PEG could modify the destabilization pH of blended micelles. As for

Nucleus Lysosome/endosome

Lysosome pH 4–5

Late endosome pH 5–6

Early endosome pH 6.5 Lysosome/endosome Nanoparticle

Tumor cells

Anticancer drug Ther. Deliv. © Future Science Group (2013)

Figure 5. Hypothetical mechanisms of the internalization of pH-sensitive nanoparticles and their acid-activated drug release. (A) Encountering the pH decrease in lysosome/ endosome, pH-sensitive nanoparticles will be destabilized and release their pH-sensitive drugs. (B) Nanoparticles may fuse with the endosome/lysosome membrane, releasing drugs into cytoplasm directly.

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Review | He, Li, An & Jiang endosomal pH (≥6) triggered drug release, PLLA-b-PEG should be utilized to as much as 40 wt% [87]. pH-sensitive endosomal vehicles with fusogenic ability

Another type of pH-sensitive vehicle comprises pH sensitivity, in addition to utilizing fusogenic lipids to assist drug escape from the endosome and lysosome to cytoplasm. Fusogenic lipids are lipids that mimic the fusion of viral envelopes with host–cell endosome membranes during viral infections [89]. Dioleoyl-phosphatidylethanolamine is the most famous fusogenic lipid, which has been utilized in liposomes to achieve efficient drug endosomal/lysosomal escape via destabilization of the membrane of the endosome/lysosome [90–93]. Sánchez et al. developed a dirhamnolipid secreted by Pseudomonas aeruginosa, which possesses a carboxylic group conferring a negative charge at neutral pH [94]. They modified dioleoyl-phosphatidylethanolamine developing a pH-sensitive liposome with fusogenic ability. Above pH 6.0, no lipid mixing was observed, while at pH 5.6 membrane fusion can be seen and lead to leakage of liposome contents. Other than phospholipids, poly(glycicol) derivatives [92] that contain carboxyl groups and bulky steric structures have been synthesized to develop pH-sensitive liposomes with fusogenic ability. Yuba et al. utilized egg yolk phosphatidylcholine and succinylated poly(glycidol) (SucPG), constructing a stable liposome [95]. The pH-dependent fusion ability of egg yolk phosphatidylcholine-SucPGs liposome was identified at various pH values. Compared with unmodified liposomes, the SucPG liposomes demonstrated an increased cellular fusion below pH 6.0, suggesting the acidic pH generated fusion ability [95]. Banerjee et al. used poly(styrene-co-maleic acid), which is known to hypercoil from a charged random coil conformation at neutral pH to a collapsed uncharged globular form at acidic pH in the endosome [96,97], anchoring distearoylphosphatidylcholine onto SMA and constructed pH-sensitive membrane fusogenic large unilamellar vesicles LUVs. The anionic amphiphile derived from Na,Nedioctanoyl lysine with a lithium counterion also showed pH-responsive membrane-lytic behavior [98]. Na,Ne-dioctanoyl lysine with a lithium counterion was chosen as a bioactive excipient in designing the pH-sensitive chitosan NPs (CS-NPs) as a promising carrier for the anticancer drug MTX. The intro release assay 1506

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demonstrated a significantly faster drug release at pH 5.4 after 1h, indicating the pH-induced drug release properties of MTX-CS-NPs. Incubated with MTX-CS-NPs, the lysosomal membrane of HeLa cells showed little or no staining by microscopic observation, which proved the membrane-lytic ability. Sanjoh et al. chose poly(N-[N-(2-aminoethyl)-2-aminoethyl] aspartamide) (pAsp-[DET]), a cationic polymer with fusogenic ability to condense pDNA and achieve the endosomal-disruption [99] . They also utilized pAsp(DET-Acetoxy [Aco]), which could turn into pAsp(DET) at endosomal/ lysosomal pH via the hydrolysis of cis-aconitic amide moiety. In summary, they used layer-bylayer technology to condense pDNA, developing pDNA/pAsp-(DET) complex, then adding the anionic polymer pAsp(DET-Aco), formulating the ternary polyplex pDNA/pAsp-(DET)/ pAsp(DET-Aco). The z potential of the ternary polyplex maintained at -40 mV at pH 7.4. The z potential increased from negative to positive at pH 5.5, indicating the pH sensitivity of such a polyplex and resulting in the exposure of fusogenic pAsp-(DET). With the help of pAsp(DET), therapeutic pDNA could be released to the cytoplasm, achieving cancer therapy. In summary, adding membrane-lytic moieties to carboxylic acid-containing materials or chargeconversional drug-delivery systems could not only promote drug efflux to endosomes/lysosomes via response to endosomal/lysosoml acidic environment, it could also promote membrane destabilization of the endosome/lysosome, achieving a much more effective drug release to cytoplasm. Conclusion Tumor pHe sensitive vehicles modified with pH-sensitive peptides as targeting moieties or achieving ligand exposure by shielding/deshielding or pop-up mechanisms are novel anticancer drug-delivery systems for the release of longstanding problem of non-specific anticancer drug delivery. Endosomal/lysosomal acidic pH-sensitive drug-delivery systems containing acid-labile chemical bonds or acid-unstable polymers can mediate high doses of anticancer drug release in cancer cells, achieving efficient drug concentrations and overcoming multiple drug resistance. pH-sensitive polymers with no toxicity and excellent biocompatibility should be investigated. Furthermore, pH-sensitive drugdelivery systems should be applied in various anticancer drugs other than doxorubicin and paclitaxel, achieving a more general platform future science group

pH-sensitive drug-delivery systems for tumor targeting for tumor chemotherapy. In future, clinical data of novel pH-sensitive tumor-targeting drug-delivery systems should also be evaluated. Future perspective Drug-delivery systems responding to stimuli, especially pH change, have been well investigated recently. As pH decrease is a phenomenon widely observed in most solid tumors, pH-sensitive vehicles provide a more general strategy for tumor-targeting therapy. Moreover, combining this with an active targeting strategy, pH-sensitive vehicles could improve the tumor-targeting specificity and provide a method to reduce the side effects in tumor therapy. Endosomal/lysosomal pH-sensitive vehicles could promote rapid drug release in tumor cells, providing an effective solution to multidrug resistance. Other than anticancer drug delivery, it is also conceivable that pH-sensitive NPs could be applied in anticancer

| Review

gene delivery and tumor diagnosis. More investigations should be designed into biodegradable polymers that could respond to pH changes in extracellular tumor tissue and endocytic pathway related organelles. Most pH-sensitive vehicles are only in the basic research phase. In the future, effective pH-sensitive tumor-targeting NPs with no apparent cyto­toxicity and systemic toxicity should be tested in clinical trials. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert t­estimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

Executive summary The pH environment in tumors „„

Induced by the Warburg effect and CO2 produced via oxidative metabolism, there is increased proton production in tumors. Combined with a decrease in proton clearance, the microenvironment in cancerous tissues appears slightly acidic.

The endosome and lysosome are critical organelles mediating drug-vehicle internalization. The acidic pH of the endosome and lysosome is essential in triggering pH-sensitive drug release. Extracellular pH-triggered tumor-targeting nanoparticles „„

„„

pH-sensitive peptides, pH-triggered ionized polymers, ligand exposure by the pop-up and shield/deshielding mechanisms are four main approaches for improving the tumor targeting properties of pHe.

„„

pH-sensitive peptides mainly include GALA, pHILP and histidine-modified cell-penetrating peptides.

„„

pH-triggered ionized polymers usually contain sulfonates, carboxylic acids and amine groups mediating charge-flip with pH decrease, achieving pH sensitivity.

„„

The shield/deshielding mechanism contains shielding units that could be removed at pHe, exposing a cationic complex or targeting ligands.

The pop-up mechanism utilizes histidine-containing polymers that could transform their configuration via pH decrease. Therefore, target ligands could be pushed outside, mediating active targeting. Intracellular pH-triggered tumor-targeting nanoparticles „„

„„

Directly conjugating anticancer drugs via acid-cleavable linkages and utilizing acid-unstable polymers are two main approaches for endosomal/lysosomal pH-sensitive drug-delivery systems.

„„

Acid-labile chemical bonds such as hydrazone bonds, cis-aconityl bonds and ether linkages have been used to directly link drugs to nanoparticles and release drugs rapidly in the endosome and lysosome environments.

„„

Acid biodegradable polymers or pH-triggered ionized polymers are used in constructing acid-unstable nanoparticles mediating drug efflux to the organelles. pH-sensitive polymers with fusion ability could promote efficient drug release to the cytoplasm.

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