Acidic Tumor Environment Research Articles

In Vitro and In Vivo Antitumor Activity of a Novel pH-Activated Polymeric Drug Delivery System for Doxorubicin

BackgroundConventional chemotherapy agent such as doxorubicin (DOX) is of limited clinical use because of its inherently low selectivity, which can lead to systemic toxicity in normal healthy tissue.MethodsA pH stimuli-sensitive conjugate based on polyethylene glycol (PEG) with covalently attachment doxorubicin via hydrazone bond (PEG-hyd-DOX) was prepared for tumor targeting delivery system. While PEG-DOX conjugates via amid bond (PEG-ami-DOX) was synthesized as control.ResultsThe synthetic conjugates were confirmed by proton nuclear magnetic resonance (NMR) spectroscopy, the release profile of DOX from PEG-hyd-DOX was acid-liable for the hydrazone linkage between DOX and PEG, led to different intracellular uptake route; intracellular accumulation of PEG-hyd-DOX was higher than PEG-ami-DOX due to its pH-triggered profile, and thereby more cytotoxicity against MCF-7, MDA-MB-231 (breast cancer models) and HepG2 (hepatocellular carcinoma model) cell lines. Following the in vitro results, we xenografted MDA-MB-231 cell onto SCID mice, PEG-hyd-DOX showed stronger antitumor efficacy than free DOX and was tumor-targeting.ConclusionsResults from these in vivo experiments were consistent with our in vitro results; suggested this pH-triggered PEG-hyd-DOX conjugate could target DOX to tumor tissues and release free drugs by acidic tumor environment, which would be potent in antitumor drug delivery.

PH-Sensitive Polymeric Micelle-Based pH Probe for Detecting and Imaging Acidic Biological Environments

To overcome the limitations of monomeric pH probes for acidic tumor environments, this study designed a mixed micelle pH probe composed of polyethylene glycol (PEG)-b-poly(L-histidine) (PHis) and PEG-b-poly(L-lactic acid) (PLLA), which is well-known as an effective antitumor drug carrier. Unlike monomeric histidine and PHis derivatives, the mixed micelles can be structurally destabilized by changes in pH, leading to a better pH sensing system in nuclear magnetic resonance (NMR) techniques. The acidic pH-induced transformation of the mixed micelles allowed pH detection and pH mapping of 0.2-0.3 pH unit differences by pH-induced "on/off"-like sensing of NMR and magnetic resonance spectroscopy. The micellar pH probes sensed pH differences in nonbiological phosphate buffer and biological buffers such as cell culture medium and rat whole blood. In addition, the pH-sensing ability of the mixed micelles was not compromised by loaded doxorubicin. In conclusion, PHis-based micelles could have potential as a tool to simultaneously treat and map the pH of solid tumors in vivo.

Surface Charge Switchable Nanoparticles Based on Zwitterionic Polymer for Enhanced Drug Delivery to Tumor

Two faced nanoparticles: A zwitterionic polymer-based nanoparticle with response to tumor acidity is developed for enhanced drug delivery to tumors. The nanoparticles are neutrally charged at physiological conditions and show prolonged circulation time; after leaking into tumor sites, in the acidic extracellular tumor environment (pH(e) ), nanoparticles are activated and become positively charged and are therefore efficiently taken up by tumor cells, leading to enhanced therapeutic effects in cancer treatment.

High-sensitivity detection of breast tumorsin vivoby use of a pH-sensitive near-infrared fluorescence probe

We investigated the potential of the pH-sensitive dye, CypHer5E, conjugated to Herceptin (pH-Her) for the sensitive detection of breast tumors in mice using noninvasive time-domain near-infrared fluorescence imaging and different methods of data analysis. First, the fluorescence properties of pH-Her were analyzed as function of pH and/or dye-to-protein ratio, and binding specificity was confirmed in cell-based assays. Subsequently, the performance of pH-Her in nude mice bearing orthotopic HER2-positive (KPL-4) and HER2-negative (MDA-MB-231) breast carcinoma xenografts was compared to that of an always-on fluorescent conjugate Alexa Fluor 647-Herceptin (Alexa-Her). Subtraction of autofluorescence and lifetime (LT)-gated image analyses were performed for background fluorescence suppression. In mice bearing HER2-positive tumors, autofluorescence subtraction together with the selective fluorescence enhancement of pH-Her solely in the tumor's acidic environment provided high contrast-to-noise ratios (CNRs). This led to an improved sensitivity of tumor detection compared to Alexa-Her. In contrast, LT-gated imaging using LTs determined in model systems did not improve tumor-detection sensitivity in vivo for either probe. In conclusion, pH-Her is suitable for sensitive in vivo monitoring of HER2-expressing breast tumors with imaging in the intensity domain and represents a promising tool for detection of weak fluorescent signals deriving from small tumors or metastases.

Abstract LB-367: Bioluminescent transposon screen identifies cancer cell specific promoters in Salmonella typhimurium

Abstract The ability of Salmonella Typhimurium to specifically localize to malignant tumors in vivo has been well documented and offers a powerful avenue for targeted anti-cancer therapies. S. Typhimurium, a common gastrointestinal pathogen, utilizes a complex array of type III secretion protein complexes, effector molecules and transcription factors during its normal pathophysiology. The expression of these genes is highly regulated in a spatiotemporal manner in response to the host environment. Akin to other host environments, such as the gastrointestinal tract and phagosomes, tumors have specific extracellular properties such as low pH and hypoxia. Identifying S. Typhimurium genes and their promoters that respond specifically to these tumor environmental properties has served as the basis for several lines of research with the hope of utilizing S. Typhimurium for anti-cancer based therapies. To identify such genes that are regulated in a tumor specific manner, we engineered a bioluminescent transposon reporter-trap to screen a S. Typhimurium library for genes specifically regulated by co-culture with malignant cells in vitro. Five previously undiscovered genes were identified (adiY, yohJ, STM1787, STM1791, and STM1793) and their promoter sequences were found to be specifically activated by the acidic microenvironment associated with cancer cells in vitro and tumors in vivo. Three of the genes (STM1787, STM1791, and STM1793) are controlled by a single promoter, which contains a recently discovered conserved tusp motif. We have uncovered that transcriptional regulation of this motif is more complex as it is sensitive to low pH in addition to hypoxia as was originally ascribed. The genes identified herein are highly expressed in an acidic pH tumor environment, but are not required for bacterial tumor targeting. Therefore, the promoters regulating these genes may be ideal candidates for utilization in therapeutic gene, pro-drug or toxin delivery studies. To address this inquiry, we utilized the cancer cell-activated STM1787 promoter sequence to regulate the expression of a toxic tumor transgene in a wild type strain of S. Typhimurium. Shiga-like toxin 2 (Stx2) is a holoenzyme produced by some strains of E.coli. It has been extensively studied for the potent translational inhibition characteristics of the A subunit in addition to the high tumor cell binding-specificity of the B subunit. Conditionally regulating the tumor-specific expression of Stx2 by STM1787's promoter resulted in striking cancer cell death in vitro, and shows promise in mouse xenografts in vivo. In conclusion, we have identified novel tumor-specific genes expressed by S. Typhimurium, identified the acidic pH-responsiveness of the tusp motif, and demonstrated that these promoters can be successfully engineered for targeted anti-cancer therapy using live S. Typhimurium. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-367. doi:1538-7445.AM2012-LB-367

Polyethyleneimine-lipid conjugate-based pH-sensitive micellar carrier for gene delivery

A low molecular weight polyethyleneimine (PEI 1.8 kDa) was modified with dioleoylphosphatidylethanolamine (PE) to form the PEI-PE conjugate investigated as a transfection vector. The optimized PEI-PE/pDNA complexes at an N/P ratio of 16 had a particle size of 225 nm, a surface charge of +31 mV, and protected the pDNA from the action of DNase I. The PEI-PE conjugate had a critical micelle concentration (CMC) of about 34 μg/ml and exhibited no toxicity compared to a high molecular weight PEI (PEI 25 kDa) as tested with B16-F10 melanoma cells. The B16-F10 cells transfected with PEI-PE/pEGFP complexes showed protein expression levels higher than with PEI-1.8 or PEI-25 vectors. Complexes prepared with YOYO 1-labeled pEGFP confirmed the enhanced delivery of the plasmid with PEI-PE compared to PEI-1.8 and PEI-25. The PEI-PE/pDNA complexes were also mixed with various amounts of micelle-forming material, polyethylene glycol (PEG)-PE to improve biocompatibility. The resulting particles exhibited a neutral surface charge, resistance to salt-induced aggregation, and good transfection activity in the presence of serum in complete media. The use of the low-pH-degradable PEG-hydrazone-PE produced particles with transfection activity sensitive to changes in pH consistent with the relatively acidic tumor environment.

Organellar (Na+, K+)/H+ exchanger NHE7 regulates cell adhesion, invasion and anchorage-independent growth of breast cancer MDA-MB-231 cells

Na+/H+ exchangers (NHEs) are a group of secondary active antiporters that regulate cellular pH, cell volume and ion homeostasis. In humans, nine isoforms (NHE1-NHE9) were identified and characterized as functional NHEs. While a growing body of evidence indicates that NHE1 generates an acidic tumor environment and thereby contributes to tumor invasion, little is known about the role of other NHE isoforms in tumor progression. NHE7 is a unique member of the NHE gene family that dynamically shuttles between the trans-Golgi network, endosomes and the plasma membrane, and regulates the luminal pH of these organelles. Here we show that NHE7-overexpression in breast cancer MDA-MB-231 cells enhances cell overlay, cell-cell adhesion, invasion, anchorage-independent tumor growth and tumor formation invivo. In contrast, NHE1-overexpression enhances tumor invasion, but it has little effect on cell adhesion or anchorage-independent tumor growth. Pathological examinations of the tumor samples derived from NHE7-overexpressing cells showed a similar appearance to aggressive tumors. Together, these results suggest that NHE7 enhances tumor progression. This is the first report to show the involvement of an organellar NHE in oncogenic processes.

The potential for blood vessel engineering of PHSRN-modified polymer: HUVEC cell affinity evaluation and integrin-mediated mechanism study

Mortality rates for ovarian cancer have declined only slightly in the past forty years since the “War on Cancer” was declared. The current standard care of ovarian cancer is still cytoredutive surgery followed by several cycles of chemotherapy. The severe adverse effect from chemotherapy drug is a leading cause for the patients to fail in long term therapy post-surgery. New nanocarriers able to minimize the premature drug release in blood circulation while releasing drug on-demand at tumor site have profound impact on the improvement of the efficacy and toxicity profile of the chemotherapeutic drugs. Here we reported a unique type of extremely long tumor retention, multi-responsive boronate crosslinked micelles (BCM) for ovarian cancer therapy. We systemically investigated the stability of BCM in serum and plasma, and their responsiveness to acidic pH and cis-diols (such as mannitol, a safe FDA approved drug for diuresis) through particle size measurement and förster resonance energy transfer (FRET) approach. Paclitaxel (PTX) loaded BCM (BCM-PTX) exhibited higher stability than non-crosslinked micelles (NCM) in the presence of plasma or serum. BCMs possessed a longer in vivo blood circulation time when compared to NCM. Furthermore, BCM could be disassembled in an acidic pH environment or by administrating mannitol, facilitating drug release in an acidic tumor environment and triggered by exogenous stimuli after drug enrichment in tumor mass. Near infra-red fluorescence (NIRF) imaging on SKOV-3 ovarian cancer mouse model demonstrated that the NIR dye DiD encapsulated BCM could preferentially accumulate in tumor site and their tumor retention was very long with still 66% remained on 12th day post injection. DiD-NCM had similar high-level uptake in tumor with DiD-BCM within the first 3 days, its accumulation, however, decreased obviously on 4th day and only 15% dye was left 12 days later. In both formulations, the dye uptake in normal organs was mostly washed away within the first 24–48 h. In in vivo tumor treatment study, PTX loaded BCM showed superior therapeutic efficacy than that of NCM and Taxol. The mice could tolerate 20 mg/kg PTX formulated in nano-formulations, which doubled the maximum tolerated dose (MTD) of Taxol. The administration of mannitol 24 h after BCM-PTX injection further improved the tumor therapeutic effect and elongated the survival time of the mice. The novel boronate-catechol crosslinked nanocarrier platform demonstrated its superior capability in targeted drug delivery, which is not only useful for ovarian cancer treatment but will also be beneficial for the therapy of many other solid tumors.

Block Copolymer Micelles for Controlled Delivery of Glycolytic Enzyme Inhibitors

To develop block copolymer micelles as an aqueous dosage form for a potent glycolytic enzyme inhibitor, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO). The micelles were prepared from poly(ethylene glycol)-poly(aspartate hydrazide) [PEG-p(HYD)] block copolymers to which 3PO was conjugated through an acid-labile hydrazone bond. The optimal micelle formulation was determined following the screening of block copolymer library modified with various aromatic and aliphatic pendant groups. Both physical drug entrapment and chemical drug conjugation methods were tested to maximize 3PO loading in the micelles during the screening. Particulate characterization showed that the PEG-p(HYD) block copolymers conjugated with 3PO (2.08∼2.21wt.%) appeared the optimal polymer micelles. Block copolymer compositions greatly affected the micelle size, which was 38nm and 259nm when 5kDa and 12kDa PEG chains were used, respectively. 3PO release from the micelles was accelerated at pH 5.0, potentiating effective drug release in acidic tumor environments. The micelles retained biological activity of 3PO, inhibiting various cancer cells (Jurkat, He-La and LLC) in concentration ranges similar to free 3PO. A novel micelle formulation for controlled delivery of 3PO was successfully prepared.

Stealth properties of poly(ethylene oxide)-based triblock copolymer micelles: A prerequisite for a pH-triggered targeting system

Evaluation of the biocompatibility of pH-triggered targeting micelles was performed with the goal of studying the effect of a poly(ethylene oxide) (PEO) coating on micelle stealth properties. Upon protonation under acidic conditions, pH-sensitive poly(2-vinylpyridine) (P2VP) blocks were stretched, exhibiting positive charges at the periphery of the micelles as well as being a model targeting unit. The polymer micelles were based on two different macromolecular architectures, an ABC miktoarm star terpolymer and an ABC linear triblock copolymer, which combined three different polymer blocks, i.e. hydrophobic poly(ε-caprolactone), PEO and P2VP. Neutral polymer micelles were formed at physiological pH. These systems were tested for their ability to avoid macrophage uptake, their complement activation and their pharmacological behavior after systemic injection in mice, as a function of their conformation (neutral or protonated). After protonation, complement activation and macrophage uptake were up to twofold higher than for neutral systems. By contrast, when P2VP blocks and the targeting unit were buried by the PEO shell at physiological pH, micelle stealth properties were improved, allowing their future systemic injection with an expected long circulation in blood. Smart systems responsive to pH were thus developed which therefore hold great promise for targeted drug delivery to an acidic tumoral environment.

Combined Multimodal Optical Imaging and Targeted Gene Silencing Using Stimuli-Transforming Nanotheragnostics

Combined diagnosis and therapy for cancer has been of great interest in medicine. Small interference RNA (siRNA)-encapsulating polyplexes were covalently coated with small gold nanoparticles (Au NPs) via acid-cleavable linkages in order to explore the possibility of achieving combined stimuli-responsive multimodal optical imaging and stimuli-enhanced gene silencing. In a mildly acidic tumor environment, Au NPs are dissociated from the siRNA-carrying polyplexes, generating various optical signal changes such as diminished scattering intensity, increased variance of Doppler frequency, and blue-shifted UV absorbance (stimuli-responsive imaging). Simultaneously, Au NP dissociation exposes the siRNA-carrying polyplex with elevated surface charge and results in enhanced cellular uptake and transfection (stimuli-enhanced therapy). In this study, the feasibility of achieving combined diagnosis and therapy for cancer (theragnostics) is demonstrated by (1) microscopic and spectrophotometric confirmation of acid-transformation of the nanoparticles, (2) reduced scattering intensity and increased variance of Doppler frequency in an acidic pH upon the nanoparticle's transformation, and (3) simultaneous optical signal changes and gene silencing in vitro under a tumor pH-mimicking condition. This novel type of stimuli-responsive nanotheragnostics will provide a new paradigm for pinpointed, multimodal, and combined imaging and therapy for cancer.

Tumor Tissue-Derived Formaldehyde and Acidic Microenvironment Synergistically Induce Bone Cancer Pain

BackgroundThere is current interest in understanding the molecular mechanisms of tumor-induced bone pain. Accumulated evidence shows that endogenous formaldehyde concentrations are elevated in the blood or urine of patients with breast, prostate or bladder cancer. These cancers are frequently associated with cancer pain especially after bone metastasis. It is well known that transient receptor potential vanilloid receptor 1 (TRPV1) participates in cancer pain. The present study aims to demonstrate that the tumor tissue-derived endogenous formaldehyde induces bone cancer pain via TRPV1 activation under tumor acidic environment.Methodology/Principal FindingsEndogenous formaldehyde concentration increased significantly in the cultured breast cancer cell lines in vitro, in the bone marrow of breast MRMT-1 bone cancer pain model in rats and in tissues from breast cancer and lung cancer patients in vivo. Low concentrations (1∼5 mM) of formaldehyde induced pain responses in rat via TRPV1 and this pain response could be significantly enhanced by pH 6.0 (mimicking the acidic tumor microenvironment). Formaldehyde at low concentrations (1 mM to 100 mM) induced a concentration-dependent increase of [Ca2+]i in the freshly isolated rat dorsal root ganglion neurons and TRPV1-transfected CHO cells. Furthermore, electrophysiological experiments showed that low concentration formaldehyde-elicited TRPV1 currents could be significantly potentiated by low pH (6.0). TRPV1 antagonists and formaldehyde scavengers attenuated bone cancer pain responses.Conclusions/SignificanceOur data suggest that cancer tissues directly secrete endogenous formaldehyde, and this formaldehyde at low concentration induces metastatic bone cancer pain through TRPV1 activation especially under tumor acidic environment.

Progress in photodynamic therapy on tumors

Photodynamic therapy (PDT) is a promising treatment on neoplastic pathologic tissues, which involves the administration of a photosensitizing agent followed by the exposure of the tissue to visible nonthermal light. Light energy is captured and transferred to other molecules resulting in the formation of short-lived energetic species, which interact with biological systems and then produce tissue damage. Photosensitizer can be taken up selectively by tumor cells because of the upregulation of low-density lipoprotein receptor-mediated endocytosis and the acidic tumor environments. In recent years, the application of PDT in the treatment of malignant lesions has increased dramatically. The first health agency approval for PDT was granted for Photofrin in Canada in 1993, and, now, it is licensed in many countries for the treatment of cancers. Although Photofrin is the most commonly used photosensitizer, it has significant side effects. Therefore, major effort has been invested in the development of new sensitizers and, to this end, many photosensitizers have been described and some are now in clinical trials.

Spectral imaging of MC540 during murine and human colon carcinoma cell differentiation.

We studied the staining pattern of merocyanine 540 (MC540) by spectral imaging of murine CT26 and human HT29 colon carcinoma cells incubated with the dye MC540. This dye, usually considered a potential membrane probe, localized mainly in the cytoplasmic vesicles of the colon carcinoma cells. However, in cells incubated in an environment similar to that of a tumor (pH 6.7), high fluorescence was detected in the nuclear membrane and nucleoli. Under these acidic conditions, resembling the Krebs effect, a population of CT26 cells displayed fluorescent plasma membranes. In differentiating cells, exhibiting cell cycle arrest at G(1)-phase and an elevated level of alkaline phosphatase, MC540 fluorescence was confined to cytoplasmic vesicles and was not detected in the plasma membrane or in the nucleoli. Cell sorting analysis of both cell types at pH 5.0 revealed higher fluorescence intensity in proliferating cells compared to differentiating cells. The fluorescence intensity of MC540-stained cells reached a maximum at pH 5.0, although the fluorescence of MC540 dye was maximal at pH 7.2. This phenomenon may result from increased binding of MC540 monomers to the cells because disaggregation of the dye with Triton X-100 produced similar results. We conclude that nucleolar localization of MC540 and an elevated fluorescence intensity can be used as indicators for proliferating cells in the characteristically acidic tumor environment. (J Histochem Cytochem 49:147-153, 2001)

Effects of extracellular pH on intracellular pH-regulation and growth in a human colon carcinoma cell-line

Mechanisms of intracellular pH (pH i) regulation seem to be involved in cellular growth and cell division. Little is known about how extracellular acidosis, known to occur in central regions of solid tumors, or alkaline conditions affect pH i regulation in colonic tumors. pH i changes in the colonic adenocarcinoma cell-line SW-620 were recorded by spectrofluorimetric monitoring of the pH-sensitive, fluorescent dye BCECF, and proliferative activity was assessed by [ 3H]thymidine uptake. Resting pH i in Hepes-buffered solution was 7.53 ± 0.01 ( n = 36). Both 1 mM amiloride and Na +-free solution inhibited pH i recovery from acidification and decreased pH i in resting cells. In HC0 3 −/CO 2-buffered media resting pH i was 7.42 ± 0.01 ( n = 36). Recovery from acidification was Na +-dependent, Cl −-independent, and only partially blocked by 1 mM amiloride. In the presence of amiloride and 200 μM H 2 DIDS pH i recovery was completely inhibited. In Na +-free solution pH i decreased from 7.44 ± 0.04 to 7.29 ± 0.03 ( n = 6) and no alkalinization was observed in Cl −-free medium. Addition of 5 μM tributyltin bromide (an anion/OH − exchange ionophore) caused pH i to decrease from 7.43 ± 0.05 to 7.17 ± 0.08 ( n = 5). The effects of pHo on steady-state pH i, pH i recovery from acidification and proliferative activity after 48 h were investigated by changing buffer [C0 2] and [HC0 3]. In general, increases in pH o between 6.7 and 7.4 increased pH i recovery, steady-state pH i and growth rates. In summary, SW-620 cells have a resting pH i > 7.4 at 25°C, which is higher than other inte cells. Acid extrusion in physiological bicarbonate media is accomplished by a pH i-sensitive Na +/H + exchanger and a pH i-insensitive Na +-HC0 3 − cotransporter, both of which are operational in control cells at the resting pH i. No evidence for activity of a Cl −/HC0 3 − exchanger was found in these cells, which could account for the high pH i observed and may explain why the cells continue to grow in acidic tumor environments.