The acidic tumor microenvironment: a target for smart cancer nano-theranostics

Abstract

The acidic tumor microenvironment (TME), which mainly results from the high glycolytic rate of tumor cells, has been characterized as a hallmark of solid tumors and found to be a pivotal factor participating in tumor progression. Recently, due to the increasing understanding of the acidic TME, it has been shown that the acidic TME could be utilized as a multifaceted target during the design of various pH-responsive nanoscale theranostic platforms for the precise diagnosis and effective treatment of cancers. In this article, we will give a focused overview on the latest progress in utilizing this characteristic acidic TME as the target of nano-theranostics to enable cancer-specific imaging and therapy. The future perspectives in the development of acidic TME-targeting nanomedicine strategies will be discussed afterwards.

tumor microenvironment, cancer targeting, nanomedicine, theranostics

INTRODUCTION

Cancer is one of leading causes of human mortality around the world [1]. The current mainstream cancer-treatment modalities (e.g. surgery, chemotherapy and radiotherapy) only show limited treatment outcomes, partly owing to the complexities and heterogeneity of tumor biology [2–4]. In the past several decades, nanomedicine has been proposed to be a promising approach to achieving improved cancer diagnosis and therapy [5]. In particular, the enhanced permeability and retention (EPR) effect, which originated from the leaky tumor vessels and lack of functional lymphatic vasculatures in solid tumors, has been playing a critical role in nanomedicine to allow those drug-loaded nanoparticles to enter and accumulate inside the tumor, offering the possibility to enhance therapeutic efficacy and reduce the side effects associated with traditional chemotherapy [6–8]. However, growing evidence has also indicated that the EPR effect, although rather significant for tumor models grown on animals, may not be so effective for tumors on real human patients, and could often vary between different patients and tumor types, and even within the same tumor at different stages over time [9]. As a result, most of those clinically used nanomedicine drugs failed to show a dramatic improvement in the treatment outcome compared to their free drug formulations, although they did obviously increase the patient’s comfort by reducing the side effects during chemotherapy [10,11].

To improve the therapeutic benefits of nanomedicine, active tumor targeting, which is achieved by covalently conjugating tumor-specific targeting moieties (e.g. antibodies, peptides, aptamers, small molecules) that recognize receptors over-expressed on tumor cells (or tumor vasculatures) onto the surface of nano-therapeutics, has been extensively explored in the past few decades [12,13]. However, owing to the significant patient-to-patient variations in tumor-specific receptor expressions, as well as the increased complexity in nanomedicine formulations when targeting ligands are involved in the nanoparticle fabrication, the active tumor-targeting strategy with drug-loaded nanoparticles has only shown limited success for their clinical translation [14–16].

Over the past decade, mounting evidence has demonstrated that the tumor microenvironment (TME), which is composed of cancer cells, stromal cells, immune cells as well as extracellular matrices, shows drastically different characteristics in angiogenesis, perfusion, oxygenation and metabolic state compared with the physiological status in normal tissues [17,18]. Unlike normal cells energized via oxidative phosphorylation, tumor cells utilize the energy produced from the oxygen-independent glycolysis for survival by adapting to insufficient tumor oxygen supply resulting from the heterogeneously distributed tumor vasculatures (also known as the Warburg effect) [19,20]. Via such oncogenic metabolisms, tumor cells would produce a large amount of lactate along with excess protons and carbon dioxide, which collectively contribute to enhanced acidification of the extracellular TME with pH often in the range of 6.5–6.8, leading to increased tumor metastasis and treatment resistance [21].

It has been demonstrated that such acidic TME could be utilized as a promising target for tumor-specific imaging and therapy, offering advantages over conventional receptor-ligand-based tumor-targeting strategies, as the reduced pH is a general characteristic for most types of solid tumors [6]. To date, with the fast advances in nanotechnology, several different catalogs of nanomaterials including both organic polymers and inorganic nanomaterials with excellent tumor acidic pH-responsive transition in their physicochemical properties (e.g. surface charge, sizes, etc.), acid-triggered cleavage of covalent bonds, as well as acid-triggered decomposition behaviors, have been widely explored for design of various different types of cancer-targeted nano-theranostics [22,23]. It has been demonstrated that such acidic TME-responsive cancer nano-theranostics could enable highly specific and efficient tumor imaging and therapy via different mechanisms including amplifying imaging signals, enhancing tumor accumulation and enabling deeper intra-tumoral penetration, under pHs (6.5–6.8) slightly lower than the physiological pH (7.4) [24]. In this review article, we will introduce up-to-date progress in the design of novel multifunctional nano-theranostics for precision cancer nanomedicine by targeting the unique acidic TME.

MOLECULAR MECHANISMS UTILIZED FOR THE DESIGN OF THE ACIDIC TME-RESPONSIVE NANO-THERANOSTICS

Due to the high rate of glycolysis and lack of functional lymphatic drainage systems, cancer cells produce large amounts of lactate, which would result in an acidic extracellular environment [20,25,26]. Although the pH difference between the TME (pH 6.5∼6.8) and normal tissue (pH 7.4) is rather small (less than 1.0 pH units), a large number of organic functional groups and inorganic materials, which exhibit dramatically different physiochemical properties in response to such a subtle pH change, have been widely employed for the fabrication of various different acidic TME-responsive cancer nano-theranostics (Table 1) [24,27]. For instance, many block co-polymers containing protonatable tertiary amine groups would show a sharp pH-dependent micellization and dissociation owing to the reversible transition of their hydrophobic and hydrophilic blocks [28]. By taking advantage of this property, various smart polymeric nano-theranostics with protonatable groups have been developed to enable efficient acidic TME-targeted release of cargoes, or to amplify the signal-to-noise (S/N) ratio for pH-responsive fluorescence imaging of tumors [29]. Besides, several catalogs of small near-infrared (NIR) dye molecules, whose protonation within the weak acidic pH would lead to drastically varied absorbance spectra, have recently been successfully integrated into nano-carriers and demonstrated to be promising candidates for ratiometric tumor pH detection and mapping [30,31].

Table 1.

A summary of acidic TME-targeted smart cancer nano-theranostics.

Another strategy different from employing protonatable functional groups is to develop pH-responsive linkers that can be efficiently cleaved under a TME-associated pH. 2,3-dimethylmaleic amide (DMMA), a widely used amino group protector that could be reversibly cleaved under a mild acidic circumstance at pH ∼6.8, has recently been widely utilized to fabricate various different acidic TME-triggered surface charge-reversible nano-therapeutics [16]. Upon tumor accumulation, such DMMA-functionalized nano-therapeutics would be rapidly converted to positively charged ones, enabling significantly elevated cellular uptake and thereby greatly improved treatment outcomes [32,33]. In addition to directly modifying those positively charged nanoparticles, several different DMMA-terminated anionic block polyethylene glycol (PEG) polymers have been successfully synthesized and exploited to functionalize the positively charged nanoparticles via self-assembly induced by electrostatic interactions, thus leading to the formation of PEG corona sheddable nano-therapeutics for acidic TME-targeted cancer therapy [34,35]. More recently, 2-propionic-3-methylmaleic anhydride (CDM), a derivative of DMMA, has also been synthesized and utilized for the fabrication of PEG corona sheddable and size-switchable nano-therapeutics for acidic TME-responsive cancer therapy [36–38].

Apart from those aforementioned organic nano-formulations, several types of inorganic nanomaterials, which could rapidly decompose in response to a mild acidic circumstance at pH ∼6.8, have also been found to be potential candidates for the fabrication of acidic TME-responsive nano-theranostics [39,40]. For instance, manganese dioxide (MnO₂), a type of metal oxide, has recently been extensively explored for acidic TME-responsive cancer imaging owing to its excellent acidity-triggered release of Mn²⁺, an efficient contrast agent for T₁-weighted magnetic resonance (MR) imaging [41]. Besides, several water-insoluble carbonates and phosphates such as calcium carbonate (CaCO₃) and calcium phosphates (Ca₃(PO₄)₂), which also show efficient tumor acidity-responsive decomposition, have been utilized to construct various tumor-specific nano-theranostics owing to their responsive release of imaging probes or anticancer drugs [39,42].

NANOPROBES FOR IMAGING OF THE ACIDIC TME

The sensitive and accurate imaging of heterogeneous tumors is essential for early diagnosis and precise treatment of cancers [43–45]. Since the tumor extracellular pH value is correlated with its glycolytic rate, evaluating pH in the extracellular TME has been extensively explored for precise tumor diagnosis [46]. To date, apart from directly using invasive microelectrodes, several other non-invasive imaging techniques based on in vivo magnetic resonance spectroscopy (MRS), chemical exchange saturation transfer (CEST) MR imaging, positron emission tomography (PET) and fluorescence imaging have also been developed for tumor pH imaging [47]. In recent years, with the rapid development of nanothechnology, the acidic TME has also been taken advantage of the unique characteristic of tumors for the fabrication of various innovative nanoprobes to amplify the S/N ratios of tumors and enable cancer-specific imaging [48]. In this section, those acidic TME-responsive nanoprobes utilized for fluorescence imaging, photoacoustic (PA) imaging and MR imaging will be introduced in detail.

Nanoprobes for pH-responsive fluorescence imaging

Fluorescence imaging is one of the mostly widely explored imaging modalities with advantages in having high sensitivity, being capable of multicolor imaging and being easy to operate [49–52]. However, for conventional tumor-targeted imaging strategies, the S/N ratios for tumors may not be optimal due to the autofluorescence background as well as non-specific distribution of imaging probes in normal tissues [50]. Recently, a series of nanoprobes featured with stimuli-responsive fluorescence ‘Turn on’ patterns have been demonstrated to be rather promising to differentiate tumors from their surrounding healthy tissues, as those imaging probes are always in a ‘Turn off’ state before encountering the specific stimuli such as the reduced pH in TME [53–58]. For instance, Gao and coworkers fabricated ultra pH-sensitive (UPS) nanoprobes to enable tumor-acidity-responsive signal amplification [59]. With the poly(ethylene glycol)-b- poly(2-(hexamethyleneimino)ethylmethacrylate) copolymer as the core material, the obtained UPS nanoprobe had a pH transition at 6.9 and a sharp pH response of fluorescence activation within 0.23 pH units [27]. With the conjugation of cyclic RGDfK peptide, a targeting moiety towards α_vβ₃ intergrin, this UPS nanoprobe showed excellent pH-responsive fluorescence activation within the tumor by as high as ∼300-fold compared to its silent state during the circulation. High S/N ratios of ∼10 were achieved over several different tumor models.

More recently, a simplified and optimized pH-sensitive nanoprobe was fabricated based the aforementioned UPS nanoprobe by the same group via removal of the cRGDfK ligand and replacing Cy5.5 dye with indocyanine green (ICG), a clinically used fluorophore approved by the Food and Drug Administration (FDA) of the USA (Fig. 1) [60]. The obtained pH-activatable ICG-encoded nanosensor (PINS) with transition pH at 6.9 showed further improved sharpness of the pH response from 0.26 to 0.15 pH units as well as enhanced fluorescence penetration depth owing to the longer wavelength of ICG. Such PINS not only enabled reliable detection of various tumors by using existing clinical cameras, but also showed great potential for real-time imaging-guided resection of established tumors and occult nodules (<1 mm³) in mouse models, leading to obviously improved long-term survival after cancer surgery. These promising results indicate that such a tumor-acidity-responsive fluorescence activation strategy holds great potential for highly specific tumor detection and reliable imaging-guided tumor resection, promising for further clinical translation.

Figure 1.

In vivo NIR fluorescence imaging-guided surgery of tumors with pH-activatable ICG-encoded nanosensor (PINS). (a) A scheme showing the binary off/on response of such PINS at a pH of 6.9. At pH <6.9, PINS dissociate into protonated, highly fluorescent unimers (on state); at pH >6.9, PINS is silent (off state). (b) A scheme showing the tumor metabolic imaging by PET with fludeoxyglucose (FDG) versus NIR fluorescence imaging with PINS. (c) severe combined immunodeficiency (SCID) mice bearing large (200 mm³) or small (10 mm³) HN5 orthotopic tumors. PINS imaging showed improved sensitivity and specificity of tumor detection over FDG-PET. Black arrowheads and blue arrowheads indicate false-positive detection of brown fat and striated muscle, respectively, in the PET images. The figure was originally published by [60] and has been approved for reuse by the Nature Publishing group.

Nanoprobes for pH-responsive photoacoustic imaging

Photoacoustic (PA) imaging, also known as optoacoustic or thermoacoustic imaging, is an emerging optical-imaging modality based on the photoacoustic effect by which the input pulsed laser light after being absorbed could be converted into heat, thereby leading to transient thermoelastic expansion and ultrasonic output [61,62]. Compared with traditional optical imaging (e.g. fluorescence imaging), PA imaging simultaneously enables both functional and structural imaging with superior spatial resolutions and greatly improved tissue-penetration depth to 5–8 cm [63,64]. To date, a large variety of tumor-targeting nano-agents with strong NIR absorption have been widely explored for tumor detection under PA imaging [63,65–68]. For accurate in vivo pH detection, our group in recent work

successfully fabricated a pH-sensitive albumin-based nanoprobe for in vivo tumor pH detection and imaging (Fig. 2) [30]. Via a sequential two-step process, two hydrophobic dyes, benzo[a]phenoxazine (BPO_x) and IR825, would induce assembly of human serum albumin (HSA) to form HSA--BOP_x--IR825 nanocomplexes, which were then cross-linked with glutaraldehyde to form C--HSA--BOP_x--IR825 nanoparticles with high stability for following in vivo applications. In this nanoprobe, BPO_x showed a pH-responsive absorbance peak at 680 nm, while IR825, whose absorbance at 825 nm was inert to pH changes, was utilized as the internal reference, collectively conferring ratiometric PA imaging for in vivo pH detection. Upon intravenous injection, C--HSA--BOP_x--IR825 could not only accurately detect the gradual acidification of the tumor microenvironment during the tumor growth, but also the instant tumor pH evolution induced by injection of external buffers. Moreover, using side-by-side comparison, we confirmed that PA imaging showed significant superiority over fluorescence imaging for more accurate semi-quantitative in vivo detection of tumor pH owing to its excellent deep-tissue-penetration capacity.

Figure 2.

In vivo ratiometric photoacoustic pH imaging. (a) A schematic illustration showing the formation of C-HAS-BPO_x-IR825 and its applications in pH sensing under both ratiometric fluorescence and photoacoustic imaging. (b) The scheme showing the protonation of BPO_x moiety under reduced pH. (c) UV-vis-NIR absorbance spectra of C-HAS-BPO_x-IR825 recorded in buffers with different pH values under 600-nm excitation. (d) PA imaging of C-HAS-BPO_x-IR825 dispersed in buffers with different pH values. (e) PA imaging of tumors with different sizes after mice were i.v. injected with C-HAS-BPO_x-IR825. Images were recorded at 680-nm and 825-nm excitations. (f) I₆₈₀/I₈₂₅ signal intensity ratios of tumors based on PA imaging data in (e). The figure was originally published by [30] and has been approved for reuse by John Wiley & Sons, Inc.

To further simplify the preparation of pH-responsive PA nanoprobes, in later work, we used a pH-responsive croconine (Croc) NIR dye to induce self-assembly of HSA without the integration of an inert reference dye. The obtained HSA-Cros nanoparticles showed pH-dependent opposite changes of optical absorbance and thus photoacoustic signals at 810 nm and 680 nm, whose ratios could be utilized to determine pH values [31]. Interestingly, such HSA-Croc nanoparticles could be employed for pH mapping of tumors, which uncovered the uneven distribution of pH within a solid tumor with further reduced pH inside the core. In other work by Pu and coworkers, a pH-activatable PA nanoprobe was prepared with the semiconducting oligomer (SO) acting as the inert PA matrix and a boron-dipyrromethene (BODIPY) dye serving as the pH-responsive PA signal enhancer, also enabling the sensitive tumor pH imaging [69]. Therefore, ratiometric photoacoustic imaging with a pH-response nanoprobe could provide a feasible solution for in vivo quantitative imaging of pH inside tumors.

Nanoprobes for pH-responsive MR imaging

MR imaging is a widely used non-invasive imaging technique with high spatial resolution and the capability of whole-body scanning [70–72]. The utility of contrast agents to enable shortening of the relaxation time of water protons has been found to be a useful strategy to increase the S/N ratios of lesions for more accurate diagnosis of those pathological diseases including cancers [73,74]. Therefore, the development of tumor-targeting MR contrast agents with amplified diagnostic signals within tumors by employing their intrinsic characteristic properties such as lower pH has attracted plenty of research interest [70,75]. To this end, various different manganese (Mn)-containing nanostructures have been prepared and found to be promising MR nanoprobes for tumor imaging by employing Mn²⁺ as an efficient T₁ contrast agent for MR imaging [76–78]. Taking advantage of the excellent pH-responsive decomposition of MnO₂, various MnO₂-based nano-theranostics including both bare MnO₂ nanostructures and MnO₂-containing nanocomposites have been found to be useful pH-responsive MR-imaging contrast agents [79–82]. For instance, Chen and coworkers in 2014 prepared 2D MnO₂ nanosheets via exfoliation from layered Na-MnO₂ materials. Such MnO₂ nanosheets showed great sensitivity to the mild acidity to break up, disintegrate and release Mn²⁺ ions, thereby contributing to gradually increased T₁-weighted MR-imaging signals within tumors. Interestingly, such MnO₂ nanosheets could also be utilized as a pH-responsive drug carrier owing to their excellent acidic TME responsiveness [41].

More recently, Kataoka and coworkers developed Mn²⁺-doped calcium phosphate (CaP) nanoparticles comprising a poly(ethylene glycol) shell (PEGMnCaP) as an excellent pH-activatable MR-imaging probe (Fig. 3) [42]. Upon tumor accumulation, the CaP scaffold would disintegrate and release Mn²⁺ ions by responding to the acidic TME, subsequently contributing to enhanced T₁-MR signals within tumors owing to the binding of Mn²⁺ ions with proteins that led to dramatically increased T₁ relaxivity of Mn²⁺. More inspiringly, such Mn²⁺-doped CaP nanoparticles could be applied for the identification of hypoxic regions within the tumor mass and detection of invisible millimeter-sized metastatic tumors due to their rapid and selective MR signal-amplification capacity in response to the reduced pH inside solid tumors.

Figure 3.

pH-activatable PEGMnCaP nanoparticle with signal-amplification capabilities for non-invasive MR imaging of tumor malignancy. (a) Schematic illustration of the hybrid structure of PEGMnCaP nanoparticles. (b) Release profiles of PEGMnCaP nanoparticle under physiological conditions at different pH values. (c) The r₁ relaxivity of PEGMnCaP nanoparticles in physiological environments at different pHs and with/without proteins (e.g. HSA). The interaction of Mn²⁺ ions released from PEGMnCaP with HSA increased the relaxivity (r₁). (d) In vivo MR images of subcutaneous C26 tumor-bearing mice pre and post intravenous injection (i.v.) of PEGMnCaP (left), Gd-DTPA (center) and PEGMn₂O₃ (right) measured with 1 T MRI. (e) A comparison of the T/N contrast ratio after the administration of PEGMnCaP, Gd-DTPA and PEGMn₂O₃. (f) and (g) 3D MR images of C26 tumors before (f) and 1 h after (g) i.v. injection of PEGMnCaP measured with 7 T MRI. The figure was originally published by [42] and has been approved for reuse by the Nature Publishing group.

Additionally, the superparamagnetic Fe₃O₄ nanoparticle, a typical T₂ contrast agent by shortening the transverse relaxation time of its surrounding water protons, has also been found to be a possible pH-responsive MR-imaging contrast agent by precisely controlling the interactions between Fe₃O₄ nanoparticles and surrounding water molecules [83,84]. Motivated by this interesting property, the Lee group and others prepared a series of pH-responsive MR-imaging nanoprobes by encapsulating Fe₃O₄ nanoparticle with pH-responsive polymers [85,86]. It was found that those Fe₃O₄ nanoparticles loaded inside the hydrophobic core of the pH-responsive polymeric micelles containing poly(ethylene glycol) (PEG)-poly(β-amino ester) (PAE) showed limited T₂ contrast ability owing to their inaccessible to surrounding water molecules [87]. However, upon tumor accumulation, those Fe₃O₄ nanoparticles loaded with PEG-PAE polymeric micelles under reduced pH would dissociate and expose the Fe₃O₄ nanoparticles to water molecules after protonation of amino groups in the PAE block, subsequently resulting in remarkably enhanced T₂ MR-imaging contrast.

NANO-THERAPEUTICS RESPONSIVE TO THE ACIDIC TME

Owing to the acidic TME (pH 6.5∼6.8) as well as the further reduced pHs inside late endosomes and lysosomes of cells (pH 5∼6), there have been numerous reports developing various types of acidic-responsive nanoscale delivery systems (NDDSs) in the past few decades [16,23,88–90]. To date, there are plenty of review articles that have comprehensively summarized the development of controllable NDDSs responsive to the intracellular acidic microenvironments [22,91–93]. Therefore, in this section, we will only review the latest progress of developing therapeutic nanoparticles targeting the acidic TME, via the mechanisms of enhancing tumor accumulation, retention or cellular uptake, under pHs only slightly lower than the normal physiological pH.

pH-responsive charge-conversion nanoparticles for cancer therapy

In the field of nanomedicine, the acidic TME has been widely explored as the target of many smart therapeutic NDDSs for improved cancer therapy [94,95]. It has been well recognized that the physiochemical properties of NDDSs showed significant impairment on their blood circulation, tumor accumulation, intra-tumoral penetration, as well as subsequent tumor cell internalization, thereby determining their treatment outcomes [96–100]. For instance, the positive surface charge of NDDSs could facilitate their effective cellular uptake and long-term retention inside the tumor, but in the meanwhile would cause them to be more rapidly removed from the blood circulation by macrophage clearance owing to strong electrostatic interactions between nanoparticles and the negatively charged cell surface [101–103]. To address this contradiction, a series of novel NDDSs with switchable surface charges, which are neutral or negative during the blood circulation but could be rapidly converted to positive after those nanoparticles reach the acidic TME, have recently been elucidated to be promising for optimized drug delivery towards tumors.

Differently from those widely explored pH liable chemical bonds (e.g. acetal, hydrazone, cis-acotinyl), which usually are responsive to the acidic pH at 5.0∼6.0 inside cell lysosomes, 2,3-dimethylmaleic amide (DMMA) linkage, as an exception, is able to be rapidly cleaved within the weakly acidic TME (pH ∼6.8) and has been utilized for fabrication of various different pH-responsive NDDSs [22,33,89,91,104–106]. In 2010, Wang and coworkers synthesized a pH-responsive nanogel by attaching DMMA onto amino groups on the surface of nanogel composed of poly (2-aminoethyl methacrylate hydrochloride) (PAMA) (Fig. 4a) [32]. It was demonstrated that such a PAMA-DMMA nanogel showed promising pH-dependent cellular internalization behaviors. After that, several different surface charge switchable NDDSs based on DMMA-modified zwitterionic polymer, nano-graphene oxide and others have been elaborately prepared and showed significantly improved therapeutic effects owing to the pH-responsive surface charge reverse and thereby dramatically enhanced cellular uptake by cancer cells [107,108]. More recently, Han and coworkers prepared dual targeted nanoparticles self-assembled from a peptide composed of alkylated protoporphyrin IX (PpIX) and DMMA-modified cationic nuclear localization sequence (NLS) peptide, with a PEG segment as a linker [109]. Upon tumor accumulation, such negatively charged PAPP-DMMA nanoparticles would rapidly convert into positively charged PAPP nanoparticles responsive to the acidic TME, exposing the cationic NLS peptide to facilitate the fast tumor cell internalization and thereby efficient nuclei-targeted delivery of PpIX conjugate. After laser exposure, such dual targeted PAPP-DMMA nanoparticles led to the most effective inhibition effect on tumor growth compared to other control groups, indicating that tumor acidity-triggered, nuclei-targeted nano-theranostics is an encouraging strategy for the design of tumor-specific nano-theranostics.

Figure 4.

DMMA-based acidic TME-responsive cancer nano-theranostics. (a) A scheme showing the preparation of drug-loaded tumor extracellular pH-responsive charge-reversal PAMA-DMMA nanogels for targeted cancer therapy. The figure was originally published by [32] and has been approved for reuse by John Wiley & Sons, Inc. (b) A scheme showing the fabrication process of pH-sensitive charge-reversible UCNPs featured with acidic TME-responsive PEG deshielding profile for tumor-targeted photodynamic therapy. The figure was originally published by [34] and has been approved for reuse by John Wiley & Sons, Inc.

Besides, by using the 2-propionic-3-methylmaleic anhydride (CDM), which has similar pH responsibility to DMMA, Sun and coworkers synthesized several different tumor-pH-liable linkage-bridged co-polymers and used them for safe and effective delivery of both small chemotherapeutic molecules (e.g. docetaxel) and biomacromolecule such as small interfering RNA (siRNA) (Fig. 5a) [36,37]. It was demonstrated that those polymeric nanoparticles showed pH-responsive detachment of PEG and elevation of surface charge, contributing to remarkably improved tumor accumulation of those nanoparticles and thereby efficient cancer treatment. Moreover, another tumor pH-sensitive linker, azidomethyl-methylmaleic anhydride, has also been synthesized and found to be a friendly method for protein cross-linking via mild click chemistry, showing effective pH-responsive release of functional proteins for cancer treatment [110,111].

Figure 5.

Acidic TME-responsive cancer nano-theranostics based on CDM linker. (a) A scheme showing the self-assembly of PEG-Dlink_m-R9-PCL into nanoparticles in aqueous solution and formation of D_m-NP_siRNA as well as their tumor acidity-targeted delivery. The figure was originally published by [37] and has been approved for reuse by ACS Publishing group. (b) A scheme showing the preparation of tumor acidic microenvironment-responsive clustered nanoparticles for improved tumor penetration and therapeutic efficacy. The figure was originally published by [38] and has been approved for reuse by the National Academy of Sciences of USA.

There are other types of functional groups that can be utilized to engineer TME pH-responsive therapeutic nanoparticles. Several protonatable groups with the pK_a at ∼6.8 have been employed to construct pH-responsive surface charge switchable NDDSs [29,112,113]. In 2014, Ling and coworkers reported the fabrication of pH-responsive magnetic nanogrenades (PMNs) via a self-assembly process of a pH-responsive block polymer and iron oxide nanoparticles [85]. Upon entry into the acidic TME, such PMNs would be pronated owing the presence of the imidazole group, exposing their positively charged surface to improve their cellular uptake. Furthermore, after being accumulated in the more acidic intracellular compartment, such PMNs would dissemble to activate its MR contrast ability, fluorescence signals as well as photodynamic effects of chlorin e6 (Ce6) conjugated with the block polymer, enabling efficient acidic TME-targeted dual modal imaging-guided tumor photodynamic treatment. Furthermore, poly(_L-histidine), another type of widely explored pH-responsive polymer, has also been extensively explored and integrated in various polymeric cancer nano-theranostics for improved cancer therapy owing to its excellent acidic TME-triggered drug release and improved tumor accumulation [114–116].

pH-responsive PEG corona sheddable nanoparticles for cancer therapy

It is known that the PEG corona around PEGylated nanoparticles would be able to make the surface of those nanoparticles more inert, reducing their tendency for cellular uptake. Although condensed PEG coating is favorable for prolonging the blood circulation half-lives of nanoparticles, heavily PEGylated NDDSs usually would show limited uptake by tumor cells, unfavorable for antitumor drug delivery. Therefore, much effort has been devoted to preparing smart NDDSs whose PEG shell is responsive to the TME pH. In 2012, Yang and coworkers demonstrated that the positively charged nanocomplexes composed of siRNA and polyethylenimine (PEI) after being coated with a layer of DMMA conjugated PEGylated polymer (mPEG₄₅-b-PAEP₇₅-Cya-DMMA, PPC-DA) [35]. It was demonstrated that such PPC-DA-modified nanocomplexes showed stable surface PEG coating under normal pH values, but would rapidly convert to a positively charged surface with detached PEG shell within the acidic TME, subsequently contributing to effective antitumor gene therapy.

In addition to polymeric nanoparticles, a similar strategy may be applied to fabricate pH-responsive inorganic nano-theranostics. In other work by our group, charge-reversible upconversion nanoparticles (UCNPs) were fabricated by coating UCNPs with a co-grafted polymer, poly (allylamine hydrochloride) (PAH) co-conjugated with PEG and DMMA, as the sheddable PEG corona (Fig. 4b) [34]. By utilizing the intrinsic upconversion luminescence of UCNPs, we found that such PAH-DMMA-PEG-modified UCNPs showed enhanced tumor retention owing to acidic TME-induced surface charge reverse and PEG deshielding, thereby leading to improved NIR-light-mediated photodynamic treatment of cancer. After that, this pH-responsive polymer has also been utilized to fabricate other acidic TME-responsive nano-theranostics, such as PAH-DMMA-PEG-encapsulated cisplatin (IV) prodrug-conjugated cationic carbon dots, for improved cancer treatment [117,118].

pH-response size-switchable or dissociable nanoparticles for cancer therapy

It is well known that the nanoparticle size is another critical parameter determining the therapeutic effects of those NDDSs via impairing their accumulation and diffusion depth within solid tumors [100,119]. It has been found that nanoparticles in the size range of 10–100 nm with appropriate surface coating to enable prolonged blood circulation may show efficient tumor accumulation owing to the EPR effect, while nanoparticles below 10 nm may be rapidly cleared via renal excretion with limited tumor retention upon systemic administration [120,121]. On the other hand, larger nanoparticles may show reduced intra-tumoral diffusion capability compared to smaller ones below 10 nm, unfavorable for drug delivery in cancer therapy [98,122,123]. Therefore, to solve this conflict issue, development of size-switchable NDDSs, which could effectively shrink from a large size suitable for efficient tumor accumulation via the EPR effect, to smaller ones responding to the TME pH so as to enable efficient intra-tumoral diffusion, has been proposed to be a possible approach to resolve this problem [123–126].

By taking advantage of the excellent tumor extracellular pH responsiveness, Huang and coworkers prepared a multistage nanovehicle-based conjugating N-(2-hydroxypropyl) methacrylamide (HPMA) polymer with DMMA, doxorubicin (DOX, a model anticancer drug) and a nuclear-homing cell-penetrating peptide (R8NLS ligand) [127]. Upon tumor accumulation, such a multistage nanovehicle with a size of ∼55 nm would be converted into DOX/R8NLS co-conjugated HPMA with the size at ∼10 nm owing to the cleavage of DMMA, to enable superior tumor penetration and cellular engulfment. After being accumulated inside the endo-/lysosomes with further reduced pH, the hydrazine bonds linking DOX with HPMA polymer would be hydrolysed, releasing much smaller conjugates of DOX and R8NLS ligand to facilitate the efficient nuclei transportation of DOX for more effective cancer treatment. Recently, Wang and coworkers prepared a tumor-acidity-responsive clustered nanoparticle (iCluster) via the molecular assembly of platinum (Pt) prodrug-conjugated poly(amidoamine)-graft-polycaprolactone (PCL-CDM-PAMAM/Pt) with PCL homopolymer and poly(ethylene glycol)-b-poly(e-caprolactone) (PEG-b-PCL) copolymer (Fig. 5b) [38]. Upon tumor accumulation, such iCluster at a size of ∼100 nm would release Pt prodrug-conjugated PAMAM dendrimer (∼5 nm) via the acidity-triggered cleavage of CDM, leading to a greatly improved intra-tumoral penetration ability of therapeutic agents and thereby improved tumor treatment outcome. Meanwhile, a similar concept has been confirmed by the same group by utilizing the ionizable tertiary amine groups, which showed rapid and sharp responsiveness to the acidic TME [128].

Additionally, several inorganic nanomaterials have also been utilized for the fabrication of such acidic TME-responsive size-switchable nanomedicine by utilizing their pH-responsive decomposition behaviors for more efficient tumor-targeted cancer therapy. For instance, our group recently successfully prepared intelligent multistage albumin-MnO₂ nanoparticles via a biomineralization process, in which HSA conjugated with Ce6 or cisplatin (IV) prodrug was used as the template to assist the growth of MnO₂ from Mn²⁺ under an alkaline condition (Fig. 6a and b) [40]. After tumor accumulation, such an albumin-MnO₂ nanoparticle at a size of ∼50 nm would be rapidly decomposed under reduced pH into individual therapeutic HSA conjugates, leading to more efficient intra-tumoral penetration. The greatly relieved tumor hypoxia owing to the MnO₂-triggered decomposition of endogenous hydrogen peroxide would be further favorable for the combined photodynamic and chemotherapy in synergistic cancer treatment.

Figure 6.

Size-switchable nano-theranostics constructed with decomposable inorganic nanomaterials for acidic TME-targeted cancer therapy. (a) A scheme showing the preparation of HSA-MnO₂-Ce6&Pt (HMCP) nanoparticles and (b) their tumor microenvironment-responsive dissociation to enable efficient intra-tumoral penetration of therapeutic albumin complexes. The figure was originally published by [40] and has been approved for reuse by John Wiley & Sons, Inc. (c) A scheme showing the preparation of Ce6(Mn)@CaCO₃-PEG and (d) its acidic TME-responsive dissociation for enhanced MR imaging and synergistic cancer therapy. The figure was originally published by [39] and has been approved for reuse by Elsevier B.V.

Calcium carbonate (CaCO₃) nanoparticles are another type of biomineralization nanostructure with rapid response to the slightly reduced pH, and have recently been demonstrated to be a promising candidate for the development of acidic TME-responsive nano-theranostics [129,130]. In recent work by our group, it was found that the PEGylated CaCO₃ nanoparticles showed efficient loading capacities for both DOX, a chemotherapy drug, and Mn²⁺ ions chelated chlorin e6 (Ce6(Mn)), a photosensitizer with T₁ contrast ability for MR imaging (Fig. 6b) [39]. Upon tumor accumulation, the resulting CaCO₃@Ce6(Mn)-PEG(DOX) nanoparticles showed pH-responsive decomposition to release small molecules of Ce6(Mn) and DOX. By utilizing the enhanced T₁ signal of Ce6(Mn), the release of DOX could be monitored under MR imaging and then contributed to effective cancer treatment via the combined chemo-photodynamic therapy owing to the efficient tumor penetration of those small molecules.

Fenton reaction, an important reaction process within biological systems, has recently been found to be a promising strategy for specific cancer treatment owing to the production of a highly toxic hydroxyl group (•OH) from the high level of endogenous H₂O₂ in the presence of ferrous ion [131]. Given the instability of ferrous ion in biological systems, amorphous iron nanoparticles (AFeNPs) were prepared and dedicated to gradually release ferrous ion in response to the mild acidic TME to trigger the Fenton reaction by taking advantage of the high level of H₂O₂ within the acidic TME [132]. It was found that such AFeNPs showed excellent pH and H₂O₂ dual-responsive cell-killing ability and could effectively inhibit tumor growth upon intra-tumoral injection.

Distinctly from those aforementioned acidic TME-responsive size-shrinkable nano-theranostics, several intelligent nanomaterials have recently been reported to be able to form large aggregates in response to such mild acidic TME, contributing to significantly improved tumor accumulation and retention [133]. In 2013, Liu and coworkers reported that zwitterionic gold nanoparticles obtained by modifying as-prepared gold nanoparticles with mixed-charge self-assembled monolayers would be stable in normal pH but instantly aggregate in response to the acidic TME, resulting in greatly elevated absorbance intensity in the NIR region [134]. Upon being i.v. injected, such zwitterionic gold nanoparticles showed obviously improved tumor accumulation than PEGylated gold nanoparticles of the same size, thereby leading to superior regression effects on tumor growth via NIR-light-irradiation-induced photothermal therapy. In recent work by Bu and Shi and their coworkers, it was demonstrated that Mo-based polyoxometalate (POM) clusters would rapidly self-assemble into larger assembles to enable efficient tumor accumulation as well as dramatically increased NIR absorbance, due to their electron structural change in response to the acidic and reductive TME [135]. Meanwhile, upon being i.v. injected, such Mo-based POM clusters with self-adaptive NIR absorbance increase would effectively inhibit the growth of tumors during NIR-laser-triggered photothermal therapy.

Considering the pivotal role of oxygen in the fast growth of tumors, more recent work by Bu and Shi and their coworkers demonstrated that magnesium silicide (MgSi) nanoparticles could work as an efficient deoxygenation agent in the acidic TME owing to the rapidly released intermediate silane (SiH₄), which would react with oxygen to form insoluble silicon oxide (SiO₂) aggregates [136] to tightly block the tumor capillaries and inhibit tumor growth.

CONCLUSION AND PERSPECTIVES

In this review, we have summarized the latest progress in utilizing the hostile acidic TME as the target to design various intelligent cancer nano-theranostics. Various types of pH-responsive nanoprobes have been developed to enable great signal amplification under slightly reduced pH within solid tumors, to realize sensitive imaging of tumors with high S/N ratios. With the help of ultra-sensitive pH-response nanoprobes, acidic TME-responsive fluorescence imaging would enable imaging-guided surgery, which would be of great potential to improve the efficacy and accuracy of current surgery techniques. However, fluorescence imaging still has limitation when used for direct detection of deep-set tumors. With improved tissue penetration compared to fluorescence imaging, PA imaging by using pH-responsive nanoprobes would allow semi-quantitative in vivo pH detection and mapping within tumors. Such a technique would be particularly suitable for in vivo pH imaging in small animal models to meet needs in basic and pre-clinical research. Distinct from optical-imaging approaches that are limited by the tissue-penetration depth of light, MR imaging as a well-established whole-body imaging technique with the help of pH-responsive MR-imaging probes would be suitable for non-invasive imaging of those deep-set tumors before surgery. Overall, by taking the acidic TME as the target, smart imaging nanoprobes with excellent pH-responsive signal amplification would be promising to enable more sensitive and accurate tumor diagnosis.

As far as nano-therapeutics are concerned, it has been found that the acidic TME-responsive surface charge reverse, PEG corona detachment and size shrinkage (or decomposition) of nanoparticles would facilitate the efficient tumor accumulation, intra-tumoral diffusion and tumor cellular uptake of therapeutics, leading to significantly improved cancer treatment. Therefore, the rational development of novel cancer-targeted nano-theranostics with sequential patterns of size switch from large to small, and surface charge reverse from neutral or slightly negative to positive within the tumor, would be more preferred for efficient tumor-targeted drug delivery. Moreover, for the translation of those interesting smart pH-responsive nano-therapeutics from bench to bedside, the formulation of those nanoscale systems should be relatively simple, reliable and with great biocompatibility, since many of those currently developed nano-theranostics may be too complicated for clinical translation.

Moreover, it is well known that the acidic TME not only plays a pivotal role in the initiation, progression and metastasis of tumors, but also participates in the induction of treatment resistance. Therefore, in addition to utilizing the acidic TME as the target for the design of novel cancer nano-theranostics for specific cancer treatment, it is also a promising strategy to develop novel cancer nano-theranostics capable of efficiently modulating the hostile acidic TME, so as to overcome the treatment resistance associated with those conventional cancer therapeutics. Moreover, a more comprehensive consideration of other biochemical characteristics of TME such as hypoxia, high interfacial pressure (IFP) and even immune-suppressive environment within tumors, which all play negative roles in cancer treatment, would be very helpful for the development of next-generation nano-theranostics to achieve precision cancer nanomedicine.

ACKNOWLEDGEMENTS

This work was supported by the National Research Programs from Ministry of Science and Technology (MOST) of China (2016YFA0201200), the National Natural Science Foundation of China (51525203), the Collaborative Innovation Center of Suzhou Nano Science and Technology and the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.