doxorubicin

487Nanomedicine (2014) 9(3), 487499 ISSN 1743-5889

Doxorubicin is a potent chemotherapeutic drug applied in the clinics for the treatment of various human cancers. It is typically administered as the hydrochloride salt or in liposomal forms, which are plagued with severe side effects. In recent years, pH-sensitive polymeric nanoparticles that are capable of retaining drug during circulation while actively releasing it at the tumor site and/or inside the target tumor cells have received an overwhelming interest for tumor-targeting cancer chemotherapy. This smart delivery approach has shown to elegantly resolve the in vivo stability versus intracellular drug release dilemma, as well as stealth versus tumor cell uptake dilemma. In this review, the concept and exciting new advances in pH-sensitive polymeric nanoparticles for doxorubicin delivery are presented and discussed.

Keywords: cancerchemotherapycancerstemcellscontrolledreleasedoxorubicinmultidrugresistancepH-sensitivepolymericnanoparticles

BackgroundDoxorubicin (DOX), also known as adria-mycin (ADR), is a potent chemotherapeutic drug applied in the clinics for the treatment of a wide range of human cancers, includ-ing Hodgkins lymphoma, leukemia, mul-tiple myeloma, breast cancer, osteosarcoma, ovarian cancer and lung cancer. Like other anthracyclines, DOX takes effect by inter-calating DNA in cancer cells and inhibit-ing macromolecular biosynthesis. DOX is typically administered intravenously as the hydrochloride salt (DOX HCl) or in liposo-mal forms. The hydrochloride salt formula-tions (available under the brand names Adri-amycin PFS, Adriamycin RDF and Rubex) that show a high activity against various solid tumors with a good therapeutic index are plagued with severe side effects, such as heart damage, typhlitis, heart arrhythmias, nausea and vomiting. Liposomal DOX (available as Doxil, Caelyx and Myocet) has shown clearly reduced cardiotoxicity as compared with the unencapsulated DOX. It is found, however, that PEGylated liposomal formula-tions might cause handfoot syndrome that

limits its dose and substitution for DOX HCl formulations.

In the past decade, stealth polymeric nanoparticles such as micelles and poly-mersomes have emerged as a most promis-ing technological platform for DOX deliv-ery [13]. Unlike liposomes that have to be modified with poly(ethylene glycol) (PEG) to obtain stealth liposomes, polymeric nanoparticles are intrinsically shielded by nonfouling polymers. Moreover, they also offer benefits of better stability, low immunogenicity and versatile structures and functions as compared with liposomes. With proper particle size and surface chemistry, polymeric nanoparticles have been shown to be able to circulate in vivo for a prolonged time, effectively alleviate adverse effects including cardiotoxicity, improve drug bioavailability and enhance drug tolerance. Notably, a few polymeric DOX prodrugs (PK1 [4] and PK2 [5]) and micellar DOX formulations (NK911 [6], and SP1049C [7]) have advanced to dif-ferent phases of clinical trials. However, despite significant progress, as well as great

Fenghua Meng1, Yinan Zhong1, Ru Cheng1, Chao Deng1 & Zhiyuan Zhong*,11BiomedicalPolymersLaboratory&JiangsuKeyLaboratoryofAdvancedFunctionalPolymerDesign&Application,CollegeofChemistry,ChemicalEngineering&MaterialsScience,SoochowUniversity,Suzhou,215123,PeoplesRepublicofChina *Authorforcorrespondence: Tel.:+8651265880098 Fax:+8651265880098 [email protected]

pH-sensitive polymeric nanoparticles for tumor-targeting doxorubicin delivery: concept and recent advances

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promise, nanoparticulate DOX formulations have met with limited success due to existing extra- and intra-cellular barriers, such as poor in vivo stabil-ity, short circulation time, modest accumulation in the tumor, inefficient uptake by tumor cells or slow intracellular drug release [810].

As an ideal drug vehicle, polymeric nanoparticles should be able to retain cargo during circulation while dumping all the drugs upon arriving at the site of action. Because DOX takes therapeutic effect by intercalating DNA, drug should be delivered and released into the nuclei of target cancer cells. In the recent several years, much research effort has been directed to the development of pH-sensitive poly-meric nanoparticles for intracellular DOX deliv-ery in that there exist natural pH gradients in the tumor microenvironment (pH 6.57.2) [11] and in the endosomal/lysosomal compartments of tumor cells (pH 4.06.5) [12]. It is interesting to note that a pH responsiveness strategy has been exploited to overcome various extra- and intra-cellular barri-ers to successful cancer chemotherapy for nanoscale DOX delivery systems (Figure 1). For example, tak-ing advantage of acidic extracellular pH (6.57.2) in the tumor compared with the normal tissues, super pH-sensitive nanoparticles have been developed to achieve accelerated drug release at the tumor site. pH-responsive nanoparticles that reverse charge or expose ligands at the outer surface (deshielding) at tumor pH have been designed to facilitate tumor cell uptake. Acid-sensitive nanoparticles that are prone to swelling, dissolution or degradation at endosomal/lysosomal pH (4.06.5) have been devised to obtain fast intracellular DOX release in tumor cells. More-over, pH-sensitive cross-linked nanoparticles have been proposed to resolve the extracellular stability and intracellular DOX release dilemma. These pH-sensitive nanoparticulate DOX formulations have demonstrated markedly improved antitumor activity in vitro and/or in vivo as compared with their pH-insensitive counterparts [13,14]. It should further be noted that a fast DOX release feature renders pH-sensitive nanoparticles also particularly appealing for treatment of multidrug-resistant (MDR) cancers. In addition to its high potency, DOX possesses unique solubility and fluorescence properties, which make it an ideal model for both hydrophobic (DOX in free base form) and hydrophilic (DOX in hydrochloride salt form) anticancer drugs. In this review, exciting new advances in pH-sensitive polymeric nanopar-ticles for DOX delivery are presented and discussed. At the end, a conclusion and future perspective on pH-sensitive nanoparticulate DOX formulations for cancer chemotherapy are given.

Endosomal/lysosomal pH-sensitive polymeric nanoparticles for active intracellular DOX deliveryPolymeric nanoparticles are usually internalized by cancer cells via endocytosis. Following endocytosis, rapid endosomal acidification occurs due to a vacuolar proton ATPase-mediated proton influx, which leads to a drop of pH levels in the endosomes to approximately 5.06.5 and 4.05.0 in the lysosomes [12]. In the past few years, this acidic pH in the endosomal/lysosomal compartments has been utilized as an effective means to trigger intracellular DOX release from acid-sensitive nanoparticles in order to enhance the therapeutic effi-cacy and reverse multidrug resistance in tumors [15]. It is worthy to note that pH-sensitive nanoparticles may also facilitate disruption of endosomes via a pro-ton sponge effect or interaction with the endosomal membrane.

pH-sensitive nanoparticles that are prone to swell-ing or dissolution at endosomal/lysosomal pH have been designed based on polymers containing pro-tonable amine groups, such as primary, secondary and tertiary amines. For instance, Kim et al. reported that DOX-loaded micelles based on poly(2-hydroxy-ethyl methacrylate)-b-poly(l-histidine) (p(HEMA)-b-p(His)) released DOX in a pH-dependent manner and induced higher growth inhibition of human colon tumor 116 human colon carcinoma cells at acidic than basic pH [16]. He et al. reported that DOX-loaded PEG-poly(l-histidine)-poly(l-lactic acid) (PEG

45-

PHis45

-PLLA82

) nanoparticles would swell and pro-mote DOX release at pH 5.0 due to protonation of the imidazole groups in the PHis block, inducing a high antitumor effect in HepG2 cells [17]. Qiu et al. demon-strated that DOX-loaded pH-sensitive micelles based on polyphosphazene-containing diisopropylamino (DPA) side groups had similar antitumor activity to free DOX against MCF-7 cells and a 1020-fold lower IC

50 than free DOX against drug-resistant MCF-7 cells

(MCF-7/ADR) [18]. The high antitumor efficacy was due to pH-triggered DOX release, as well as enhanced endosomal escape by the proton sponge effect of DPA moieties. Zhou et al. reported that N-deoxycholic acid-O, N-hydroxyethylation chitosan micelles modified with octreotide-PEG-deoxycholic acid exhibited pH-dependent DOX release, efficient uptake by MCF-7 cells (overexpressing somatostatin receptors) via recep-tor-mediated endocytosis and enhanced antitumor efficacy as compared with nontargeting N-deoxycholic acid-O, N-hydroxyethylation chitosan micelles in nude mice bearing MCF-7 cancer xenografts [19].

pH-sensitive nanoparticles have also been developed by incorporating acid-cleavable bonds such as hydra-zone, acetal, imine and oxime bonds onto polymer main

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or side chains. For example, van Hest et al. reported that polymersomes based on PEG-b-polybutadiene connected by a hydrazone bond displayed a strong pH-dependent colloidal stability due to pH-induced shed-ding of PEG mantles [20]. Yang et al. found that the intervening benzoic-imine linker in PEG-C18 block copolymer micelles exhibited pH-dependent progres-

sive hydrolysis behavior [21]. The imine linker, while stable at physiological pH, was partially hydrolyzed to present positive surface charge at tumor pH, facilitat-ing tumor cell uptake, and completely hydrolyzed at endosomal pH, resulting in micelle dissociation, fast intracellular DOX release and effective membrane disruption. Zhu et al. reported that DOX release from


pH-sensitive polymeric nanoparticles for tumor-targeting doxorubicin delivery: concept & recent advances Review

Tumor tissue

Tumor cell

DOX Negative/neutral surface Positive charge Targeting ligand (TAT, biotin)Protecting moiety Acid-cleavable bonds: hydrazone,

acetal, cis-acotinyl, imine and oxime

Cytoplasm

Nucleus

pH 4.06.5

+ +++

++ +

+

+

++

++

+++++

+++ +

Endosome/lysosome

v

vi

pH 6.57.2

i

iiiiv

ii

Nanomedicine Future Medicine Ltd (2014)

Figure 1. Design and development of pH-sensitive polymeric nanoparticles for doxorubicin delivery. (A) Tumor extracellular pH-sensitive nanoparticles to facilitate tumor cell uptake and/or drug release at the tumor site. (i) Tumor pH-triggered surface charge conversion (i.e., from negative or neutral to positive charge); (ii) tumor pH-triggered pop-up of specific ligands; (iii) tumor pH-triggered deshielding of CPP from the nanoparticle surface; and (iv) tumor pH-triggered nanoparticle dissociation and DOX release. (B) Endosomal/lysosomal pH-sensitive nanoparticles to accomplish fast intracellular drug release and disruption of endosomes. (v) Endosomal/lysosomal pH-triggered swelling and dissolution of nanoparticles; and (vi) endosomal/lysosomal pH-triggered degradation of nanoparticles. DOX: Doxorubicin; TAT: Trans-activator of transcription.


biodegradable micelles self-assembled from oxime-tethered PEG-PCL (poly(-caprolactone)) block copolymers was significantly accelerated at pH 5.0 as compared with physiological pH, resulting in a high anticancer efficacy in HeLa cells [22]. Along with reduc-tion-sensitive shell-sheddable micelles and polymer-somes [2325], triggered shell-shedding has appeared as a most straightforward and effective approach to achiev-ing efficient intracellular drug release. Recently, we discovered that core-cross-linked polypeptide micelles based on lipoic acid and cis-1,2-cyclohexanedicarbox-ylic acid (CCA)-conjugated PEG-poly(l-lysine) block copolymer had enhanced DOX loading at neutral pH due to the presence of ionic and hydrogen bonding interactions with CCA, but an accelerated drug release at endosomal pH owing to cleavage of the acidic-labile amide bond between CCA and PLL [26]. Interestingly, these dual-bioresponsive micelles showed more effi-cient nucleus delivery of DOX and thereby better anti-tumor activity than their pH-insensitive counterparts. Frchet et al. disclosed that micelles formed from PEO (poly(ethylene oxide))-dendritic polyester copolymer with acid-labile acetal groups on the core-forming den-drimer periphery exhibited high pH sensitivity [27]. The in vitro release studies showed that DOX release was highly pH dependent. Inspired by the work of Frchet, we have prepared pH-sensitive degradable micelles and polymersomes based on acetal-containing polycarbon-ate, poly(2,4,6-trimethoxybenzylidenepentaerythritol carbonate), as a hydrophobe [28,29]. The in vitro drug release studies showed that DOX and DOX HCl were released in a pH-dependent manner from micelles and polymersomes, respectively. In a following study, pH-sensitive degradable chimeric polymersomes were developed from PEG-poly(2,4,6-trimethoxybenzyli-dene-1,1,1-tris (hydroxymethyl) ethanemethacrylate)-poly(acrylic acid) for efficient loading of DOX HCl into the interior of polymersomes at neutral pH via the electrostatic/hydrogen bonding interactions, as well as triggered drug release at pH 5.4 through acetal hydrolysis (Figure 2) [30]. DOX HCl-loaded chime-ric polymersomes revealed a high antitumor activity comparable with free DOX HCl in HeLa cells.

The development of endosomal pH-activatable mac-romolecular prodrugs and prodrug nanoparticles with DOX covalently conjugated to the polymer chains via a cleavable hydrazone or cis-aconityl bond represents another interesting approach to pH-sensitive DOX delivery [31]. As from the 1980s, polymeric prodrugs have received tremendous interest for cancer chemo-therapy. Unlike self-assembled nanovehicles that tend to dissociate and release encapsulated drug upon intrave-nous administration, polymeric prodrugs are generally stable in blood circulation and may effectively prevent

premature drug release as a result of covalent linking of the drug to the carrier. We found that pH-sensitive PEO-g-DOX prodrugs with DOX linking to the PEO main chain via a hydrazone bond while sufficiently sta-ble at pH 7.4 were readily activatable at endosomal pH [32]. Haag et al. reported that DOX prodrugs based on dendritic polyglycerol via a cleavable hydrazone bond and with PEG shells exhibited high drug loading, good water solubility, pH-dependent drug release behavior and improved antitumor efficacy over free DOX in an ovarian xenograft tumor model (A2780) [33]. In a recent study, immunoprodrugs were developed from a poly-glycerolDOXPEG construct by conjugating a scFv antibody to the terminal of the PEG shell using SNAP-Tag (human DNA-repair enzyme O-6-alkylguanine DNA alkyltransferase) technology [34]. These immu-noprodrugs exhibited pronounced targetability and specific toxicity toward EGF receptor-overexpressing cancer cells, such as A431, MDAMB-468 and Panc-1 cells. The current polymeric prodrugs are usually based on relatively low molecular weight (


exhibited a much higher r2 relaxivity value than Feri-

dex (a commercial superparamagnetic iron oxide-based T

2 contrast agent), released DOX under mildly acidic

pH and caused higher cytotoxicity than folate (FA)-free vesicles [42]. Gu et al. reported that pH-sensitive nanoparticles formed from PEGylated peptide den-dronDOX conjugates with 14.0 wt.% drug loading displayed much faster DOX release at pH 5.0 than 7.4 due to acid cleavage of the hydrazone bonds [43]. The in vivo studies showed that these DOX nanoparticles caused no significant systemic toxicity, but had strong antitumor activity in the 4T1 breast tumor model. It should be noted, nevertheless, that DOX release from these prodrug nanoparticles is often slow and incom-plete ( MnPAA)Amphiphillic asymmetric triblock copolymer

Water-soluble triblock copolymer(complete acetal hydrolysis)

Self-assembly

DOX-HCl DOX-HCl-loaded acid-labilechimeric polymersome

pH-sensitive acetal degradation, swellingof polymersome and drug release

pH 5.0

pH 5.0

pH 5.0

Figure 2. pH-sensitive degradable chimeric polymersomes based on asymmetric poly(ethylene glycol)-poly(2,4,6-trimethoxybenzylidene-1,1,1-tris (hydroxymethyl) ethanemethacrylate)-poly(acrylic acid) triblock copolymers (poly[ethylene glycol] block longer than poly[acrylic acid] block) for active loading, as well as endosomal pH-triggered release of doxorubicinhydrochloride salt. DOX HCl: Doxorubicinhydrochloride salt; PAA: Poly(acrylic acid); PEG: Poly(ethylene glycol); PTTMA: Poly(2,4,6-trimethoxybenzylidene-1,1,1-tris [(hydroxymethyl] ethanemethacrylate). Reproduced with permission from [30].


It is interesting to note that INNO-206 has already been advanced to Phase II clinical studies [45].

The rapid intracellular drug release also renders pH-sensitive DOX nanoparticles effective against MDR cancer cells and in vivo. For example, Nagasaki et al. reported that DOX-loaded PEGylated nanogels con-taining a pH-sensitive polyamine core exhibited supe-rior antitumor activity against drug-resistant human hepatoma HuH-7 cells to their free DOX and pH-insensitive counterparts [47]. The pH-sensitive DOX-loaded PEG-poly(4-vinylbenzylphosphonate) nanopar-ticles displayed much higher cytotoxic activity against both P-gp- and MRP1-overexpressing cells than free DOX [48]. Wang et al. found that gold nanoparticles with surface-tethered DOX via a PEG spacer through a pH-sensitive hydrazone linkage achieved enhanced drug accumulation and retention in multidrug-resis-tant MCF-7/ADR cancer cells, resulting in effective reversal of multidrug resistance [49]. Qiu et al. demon-strated that DOX-loaded pH-sensitive micelles based on polyphosphazene-containing DPA side groups caused a 1020-fold lower IC

50 than free DOX against

drug-resistant MCF-7/ADR cells [18]. Lavasanifar and Xiong reported that tumor-targeting micellar nanopar-ticles based on PEO-PCL block copolymers with PCL end-functionalized using either a short polyamine for siRNA complexation or a DOX molecule through a pH-sensitive hydrazone linkage were able to simultane-ously deliver DOX and siRNA against P-gp expression into multidrug-resistant MDA-MB-435 human tumor models, leading to effective reversal of drug resistance [50]. Bae et al. disclosed that DOX-loaded micelles based on PEG-P(His-co-phenylalanine) and PEG-PLA-folate block copolymers, which targeted to folate receptors and early endosomal pH, effectively sup-pressed the growth of MDR ovarian tumor xenograft in mice for at least 50 days without weight loss [14].

Tumor pH-sensitive polymeric nanoparticles for DOX deliveryThe cancerous tissue is slightly acidic with pH values ranging from 6.5 to 7.2 due to a combination of ele-vated aerobic glycolysis and reduced blood flow. In the past few years, super pH-sensitive nanoparticles that are sufficiently stable at a physiological pH of 7.4 but are prone to deshielding, which thereby facilitate tumor cell uptake or swelling, thereby triggering drug release, have been developed for tumor-targeting drug release [5153]. Tumor extracellular pH-sensitive nanoparticles are typically designed based on polymers with pK

a in

the range of 6.5 to 7.2, such as poly(l-histidine) (PHis) and poly(-amino esters) (PAE). Bae et al. prepared ultra pH-sensitive micelles based on PEG-PHis and PEG-PLA block copolymers that were fairly stable at

pH 7.07.4, whereas they were disassembled at a pH of 6.67.2. Notably, folate-decorated DOX-loaded ultra pH-sensitive micelles showed a high cytotoxicity to drug-resistant MCF-7 cells in vitro and in vivo [54]. Lee et al. discovered that pH-responsive PEG-PAE micelles exhibited a sharp micellization/demicellization transi-tion at pH 6.46.8 [55]. DOX-loaded PEG-PAE micelles afforded enhanced tumor growth inhibition and pro-longed survival of B16F10 tumor-bearing mice as com-pared with free DOX. In a subsequent study, tumor-targeting pH-responsive micelles were constructed from PEG-PAE and AP (CRKRLDRN)-PEG-PLA [56]. The in vivo studies in mice bearing human breast MDA-MB231 tumor (overexpressing AP-specific IL-4 receptors) showed that DOX-loaded targeting micelles had better tumor accumulation and in vivo therapeu-tic efficacy than their nontargeting counterparts and free DOX. Poly(N-(3-diethylamino)propyl isothiocy-anato-l-lysine)-PEG-PLLA triblock copolymer formed flower-like micelles that were stable at pH 7.4 but dis-integrated at pH


tumor targeting, which is an appealing alternative to cell-specific active targeting.

Tumor pH-triggered pop-up or deshielding of CPP from the nanoparticle surface has been proposed to facilitate translocation of nanoparticles into tumor cells. Bae et al. reported that micelles self-assembled from PLLA-PEG-PHis-TAT and PEG-PHis copoly-mers hid TAT at physiological pH, but exposed it at a slightly acidic tumor extracellular pH [61]. More-over, the micelle core was designed to disintegrate and quickly release DOX at endosomal pH. These super pH-sensitive nanoparticles augmented DOX potency in various wild and multidrug-resistant cell lines with a 3.88.8-fold lower IC

50 than free DOX. The in vivo

studies in nude mice bearing either drug-sensitive or -resistant human xenografts all showed significant tumor regression with minimum weight loss. pH-sensi-tive micelles with reversibly shielded TAT ligands were also developed from TAT-PEG-PLLA and pH-sensitive PEG-poly(methacryloyl sulfadimethoxine) diblock copolymer, in which anionic poly(methacryloyl sul-fadimethoxine) complexed with cationic TAT of the micelles at physiological pH to yield stealth micelles [62]. However, at pH 6.6, TAT would be exposed on the micelle surfaces, leading to significantly higher cellular uptake as compared with pH 7.4, supporting shielding of TAT-PEG-PLLA micelles at normal pH and deshielding at tumor pH. Very recently, Shen et al. modified the TAT lysine residues in TAT-PEG-PCL micelles using pH-sensitive succinyl amides to inhibit nonspecific interactions of TAT in the bloodstream (Figure 3) [63]. In acidic tumor or endosomal/lysosomal compartments, succinyl amides would be quickly hydrolyzed to fully restore TATs functions. These pH-sensitive targeting micelles achieved long circulation in the blood and efficiently accumulated and delivered DOX to tumor tissues, giving rise to high antitumor activity and low cardiotoxicity.

In addition to TAT ligand, tumor pH-triggered pop-up of specific ligands (e.g., biotin) has been inves-tigated to achieve quick tumor cell uptake via receptor-mediated endocytosis. For instance, Bae et al. reported that pH-sensitive micelles based on PEG-PHis and biotin-PHis-PEG-PLA block copolymers were shielded by PEG at pH 7.4 with biotins hiding at the coreshell interface [64]. At tumor pH (pH 7.0), PHis became water soluble, exposing biotin on the micelle surface and facilitating cell uptake via receptor-mediated endocytosis. Notably, these micelles also exhibited pH-dependent dissociation and enhanced DOX release at endosomal pH. Recently, Li et al. prepared pH-sensi-tive nano-flower micelles that exhibited a half-open state to expose biotin for efficient cellular uptake at tumor pH due to acidic cleavage of the benzoicimine

bond and fully bloomed to release DOX at endosomal pH owing to cleavage of hydrazone bonds [65].

pH-sensitive cross-linked polymeric nanoparticles to resolve the extracellular stability and intracellular DOX release dilemmaOne practical challenge with nanoparticulate drug for-mulations is their low in vivo stability due to large volume dilution and/or interactions with cells and biomolecules present in the blood, which often leads to premature drug release, aggregation and diminished drug targetability [66,67]. The chemical cross-linking of nanoparticles, how-ever, can improve their stability results in usually reduced drug efficacy due to prohibited drug release at the target sites. pH-sensitive cross-linked nanoparticles have been proposed to resolve the extracellular stability and intra-cellular drug release dilemma [68]. Lee et al. reported that ketal cross-linked micelles based on PEG-PAsp-poly(l-phenylalanine) had improved stability against micelle-disrupting sodium dodecyl sulfate surfactant [69]. The release of DOX was rapid at endosomal pH as compared with physiological pH due to reversal of cross-linking via acidic degradation of ketal linkages (ketal hydrolysis half-life: 0.7 h at pH 5.0 vs 52 h at pH 7.4). The confo-cal observations showed that ketal-cross-linked micelles could efficiently release DOX in endosomes as well as into the nuclei of MCF-7 cells over 5 h. In a following study, PEG-PAsp-poly(l-phenylalanine) micelles were stabilized by calcium phosphate (CaP) that grew in the anionic PAsp shell domains (Figure 4) [70]. CaP-stabilized micelles displayed good serum stability. The release of DOX from CaP-stabilized micelles was inhibited at pH 7.4 but significantly enhanced at pH 4.5 due to rapid dissolution of CaP mineral layers. The in vivo studies in MDA-MB231 tumor-bearing mice showed that DOX-loaded CaP-stabilized micelles exhibited prolonged circulation, enhanced tumor specificity and better therapeutic efficacy compared with free DOX and non-stabilized controls. Zeng et al. discovered that pH-sen-sitive micelles assembled from P[PEGMA-b-(DEMA-co-APMA)]-FA that contains adenine (A) and tertiary amine moieties in the hydrophobic block, and FA target-ing ligand at the terminal of hydrophilic block, could be cross-linked using uracil-(CH2)6-uracil via A-U nucleo-base pairing based on complementary multiple hydrogen bonding at neutral pH [71]. These cross-linked micelles, while stable at physiological pH, were dissociated under acidic pH due to protonation of tertiary amines (pK

a

6.47.0) and disruption of the adenineuracil nucleo-base pairing. DOX-loaded micelles were preferentially taken up by folate receptor-positive cancer cells. Shuai et al. prepared pH-sensitive interlayer-cross-linked micelles from mPEG-PAsp(MEA)-PAsp(DIP)) triblock copoly-



494 Nanomedicine (2014) 9(3) future science group


CHNH

CH2 CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CO

NH

NHCO

C

OH

O

C

O

Arg ArgArgArgArgCys NH

CH

C

CO

OH

C

O

NH

CH C

O

G ly Tyr

O

CH2

CH2

CH2

CH2

CH2

CH2

NH

C

CO

OH

O

CHNH

CH2 CH2

CH2

CH2

CH2

CH2

CO

NH2

NH2

C

O

Arg ArgArgArgArgCys NH

CH C

O

NH

CH C

O

G ly Tyr

CH2

CH2

CH2

CH2

NH2

= aTAT

= TAT

Amidization(deactivation)

Acidic pH(activation)

Tumor tissue

Tumor cell

Lysosome(pH 45)

NPC

Acidic extracellular fluidPore

Blood

Figure 3. Acid-active cell-penetrating peptides for in vivo tumor-targeted drug delivery. (A) Deactivation of TAT in the blood compartment and its activation in the tumor interstitium or cells for in vivo tumor-targeted drug delivery. The amines of the lysine residues of TAT are amidized to inhibit its nonspecific interactions in the blood compartment without affecting the nanocarriers stealth properties. Once the nanocarrier extravasates into tumor tissue through highly permeable blood vessels via the enhanced permeability and retention effect, these amides are hydrolyzed, regenerating the pristine functioning CPP in the acidic tumor extracellular fluids (pH


mers, which were stable and free of drug leakage at neu-tral pH [72]. The release of DOX was, however, acceler-ated at pH 5.0. The in vivo studies in nude mice bearing the Bel-7402 xenograft demonstrated reduced premature drug release in blood circulation and better therapeutic effects than DOX-loaded PEG-PCL micelles and free DOX. Hennink et al. reported that core-cross-linked DOX prodrug micelles based on mPEG-b-p(HPMAm-Lac

n) released drug completely within 24 h at pH 5 and

37C [73]. In comparison, only approximately 5% DOX was released at pH 7.4 under otherwise the same condi-tions. These cross-linked DOX prodrug micelles demon-strated better antitumor activity than free DOX in mice bearing B16F10 melanoma carcinoma.

Conclusion & future perspectiveThe past few years have witnessed remarkable advances in pH-sensitive nanoparticles for tumor-targeting DOX delivery. The pH-responsiveness has been employed as an elegant and unique strategy to over-come various extracellular and intracellular barriers to successful cancer chemotherapy for nanoscale DOX delivery systems. For example:

Tumor extracellular pH-sensitive nanoparticles have been designed to achieve accelerated drug release at the tumor site;

pH-responsive nanoparticles that reverse surface charge or expose ligands such as TAT and biotin at



SO3K

N+N

O

KO3S

COCHNHNHOCH2CH2CH2 CH2

CH3 113

C

O

O

12COCHNH

23

Endocytosis

Nucleus

Extracellular pH 7.4Ca2+ 2 mM

Inorganic CaP layer

Anionic domain

Self-assemblyPEG PPhe

Cy 5.5

PAsp

DOX PO43-

Ca2+

CaP layerdissolution

DOX release

Figure 4. Shell-specific calcium phosphate mineralization of doxorubicin-loaded poly(ethylene glycol)-poly(l-aspartic acid)-poly(l-phenylalanine) micelles and triggered release of doxorubicin by acidic intracellular compartments. CaP: Calcium phosphate; Cy: Cyanine; DOX: Doxorubicin; PEG: Poly(ethylene glycol); PAsp: Poly(l-aspartic acid); PPhe: Poly(l-phenylalanine).Reproduced with permission from [70].


the outer surface (deshielding) at tumor extracel-lular pH have been developed to facilitate tumor cell uptake without compromising their long circulation time. This pH-triggered deshielding approach offers an indispensable alternative to cell-specific active targeting of solid tumors (based on, e.g., antibodies, peptides and aptamers, among others);

Acid-sensitive nanoparticles that are prone to swell-ing, dissolution or degradation at endosomal/lyso-somal pH (4.06.5) have been devised to obtain fast intracellular DOX release in the tumor cells. To further enhance their in vivo stability, acid-sensitive nanoparticles can also be chemically cross-linked (via either degradable or nondegradable cross-links);

pH-sensitive nanoparticles have been shown to facili-tate disruption of endosomes via the proton sponge effect and/or active interactions with endosomal membranes (caused by deshielding, and low molecular weight degradative products, among others);

pH-sensitive cross-linking of nanoparticles has been utilized not only to enhance their in vivo sta-bility, inhibit premature drug release, prolong drug circulation time and improve drug accumulation at the tumor site, but also to maintain fast drug release inside target tumor cells due to cleavage of cross-links under endosomal/lysosomal compartments;

Dual pH-sensitive nanoparticles (i.e., one sensitive to tumor pH and the other to endosomal/lysosomal pH) have been designed to accomplish prolonged circulation time and efficient tumor cell uptake, as well as fast intracellular drug release. pH-sensitive nanoparticulate DOX formulations have dem-onstrated superior in vitro and in vivo antitumor activity to free DOX and pH-insensitive controls. Interestingly, several studies have shown that DOX-loaded pH-sensitive nanoparticles are particularly effective for the treatment of MDR cancers.

It should be noted that although not included in this review, dual- and multi-stimuli-sensitive nanoparticles that respond to pH and other stimuli (e.g., redox, enzyme and near infrared, among others), either simultaneously at the same location or in a sequential manner in differ-ent compartments, have been developed to offer unprec-edented control over DOX delivery and release, leading to superior in vitro and/or in vivo anticancer potency [74].

In spite of significant progress in the development of pH-sensitive nanoparticles for DOX delivery, most are proof-of-concept studies and none have advanced to clin-ical evaluations. The first bottleneck with these smart

delivery systems, like other sophisticated anticancer drug delivery systems, is that they are usually based on innovative polymers that are difficult to obtain approval for from the authorities for medical and pharmaceutical uses. In the future, effort should be directed to advance-ment of pH-sensitive nanoparticles from well-accepted biomedical materials such as biodegradable polyesters, polycarbonates, polypeptides, PEG and dextran, among others. It should be noted that DOX-loaded pH-sensi-tive nanoparticles, although displaying better in vitro and in vivo antitumor activity as compared with free DOX and pH-insensitive counterparts, are far from optimal in terms of therapeutic performance. Given the fact that pH differences between tumor tissues or endo-somal/lysosomal compartments and normal tissues are small, pH response is often found to be slow (ranging from several hours to a few days), which results in only partial DOX release at the tumor site or inside the tumor cells. In order to achieve maximum therapeutic effects, however, drugs should preferentially be dumped upon arriving at the target site. To this end, nanoparticulate DOX formulations with super pH sensitivity should be developed for more effective cancer chemotherapy. Moreover, it should be noted that the presence of mul-tidrug resistance in cancer cells and cancer stem cells is responsible for the failure of many clinical treatments using either traditional drug formulations or nano-medicines [75]. Hence, it is of the utmost importance to invent pH-sensitive nanoparticulate DOX formulations that can reverse multidrug resistance in cancer cells and effectively kill cancer stem cells. In the future, efforts should be directed to the development of robust, tumor-targeting and super pH-sensitive nanoparticles based on well-established biocompatible materials for efficient delivery and release of DOX into solid tumor cells, as well as cancer stem cells. We are convinced that based on pH-sensitive nanoparticles, innovative and smart DOX nanomedicines may be developed for safe, effi-cient and targeted cancer therapy.

Financial & competing interests disclosureThis work is financially supported by research grants fromthe National Natural Science Foundation of China (NSFC51003070, 51103093, 51173126, 51273137 and 51273139),the National Science Fund for Distinguished Young Schol-ars (NSFC 51225302) and a project funded by the PriorityAcademic Program Development (PAPD) of Jiangsu HigherEducation Institutions. The authors have no other relevantaffiliationsorfinancial involvementwithanyorganizationorentitywithafinancialinterestinorfinancialconflictwiththesubjectmatterormaterialsdiscussedinthemanuscriptapartfromthosedisclosed.

Nowritingassistancewasutilizedintheproductionofthismanuscript.




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Executive summary

Tumor extracellular pH-sensitive nanoparticles pH-responsive nanoparticles that reverse surface charge or expose ligands such as TAT and biotin at the

outer surface (deshielding) at tumor pH have been developed to facilitate tumor cell uptake, without compromising their long circulation time.

Tumor pH-sensitive nanoparticles have been designed to achieve accelerated drug release at the tumor site.

Endosomal/lysosomal pH-sensitive nanoparticles Acid-sensitive nanoparticles that are prone to swelling, dissolution or degradation at endosomal/lysosomal pH

have been devised to obtain fast intracellular doxorubicin release in the tumor cells.

pH-sensitive nanoparticles have been shown to facilitate disruption of endosomes via the proton sponge effect and/or active interactions with endosomal membranes.

pH-sensitive cross-linking of nanoparticles has been utilized not only to enhance their in vivo stability and improve drug accumulation at the tumor site, but also to maintain fast drug release inside target tumor cells.

Doxorubicin-loaded pH-sensitive nanoparticles are particularly effective for the treatment of multi-drug resistant cancers.

Dual pH-sensitive nanoparticles Dual pH-sensitive nanoparticles have been designed to accomplish a prolonged circulation time and efficient

tumor cell uptake, as well as fast intracellular drug release.


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