Acidic Extracellular Environment Research Articles

Chemo-Mechanical Modulation of Cell Motions Using DNA Nanosprings.

Cell motions such as migration and change in cellular morphology are essential activities for multicellular organism in response to environmental stimuli. These activities are a result of coordinated clustering/declustering of integrin molecules at the cell membrane. Here, we prepared DNA origami nanosprings to modulate cell motions by targeting the clustering of integrin molecules. Each nanospring was modified with arginyl-glycyl-aspartic acid (RGD) domains with a spacing such that when the nanospring is coiled, the RGD ligands trigger the clustering of integrin molecules, which changes cell motions. The coiling or uncoiling of the nanospring is controlled, respectively, by the formation or dissolution of an i-motif structure between neighboring piers in the DNA origami nanodevice. At slightly acidic pH (<6.5), the folding of the i-motif leads to the coiling of the nanospring, which inhibits the motion of HeLa cells. At neutrality (pH 7.4), the unfolding of the i-motif allows cells to resume mechanical movement as the nanospring becomes uncoiled. We anticipate that this pH-responsive DNA nanoassembly is valuable to inhibit the migration of metastatic cancer cells in acidic extracellular environment. Such a chemo-mechanical modulation provides a new mechanism for cells to mechanically respond to endogenous chemical cues.

The pH-triggered polyglutamate brush co-delivery of MDR1 and survivin-targeting siRNAs efficiently overcomes multi-drug resistance of NSCLC

Multi-drug resistance (MDR) is one of the major challenges in the successful chemotherapy of non-small cell lung cancer (NSCLC). Although RNA interference (RNAi) has been widely used to silence resistance-related genes, the effect remains unsatisfactory. In this study, we attempted to overcome MDR of NSCLC by simultaneously interfering with two RNAs that have different functions. A new pH-triggered polyglutamate brush polymer dimethylmaleic anhydride-poly(ethyleneglycol) monomethyl ether-b-polyglutamate-g-spermine (DMA-mPEG-b-PG-g-spermine, DPPGS) was designed and synthesized. The DPPGS/small interfering RNA (siRNA) complex nanoparticles (DPPGSN) were prepared. The results demonstrated that DPPGSN could be transformed from a negatively charged form into a positively charged form in the slightly acidic tumor extracellular environment. The siRNA targeting MDR1 mRNA (siMDR1) and siRNA targeting survivin mRNA (siSurvivin) could be efficiently co-delivered by DPPGS to simultaneously interfere with two genes (p < 0.01). Furthermore, DPPGS co-delivery of siMDR1 and siSurvivin lowered the IC50 value of cisplatin (DDP) in A549/DDP (p < 0.01) cells and increased the apoptosis rate of the cells (p < 0.01). Therefore, co-delivery of siMDR1 and siSurvivin using DPPGS would be a promising approach for overcoming MDR of NSCLC.

The control of acidity in tumor cells: a biophysical model

Acidosis of the tumor microenvironment leads to cancer invasion, progression and resistance to therapies. We present a biophysical model that describes how tumor cells regulate intracellular and extracellular acidity while they grow in a microenvironment characterized by increasing acidity and hypoxia. The model takes into account the dynamic interplay between glucose and hbox {O}_2 consumption with lactate and hbox {CO}_2 production and connects these processes to hbox {H}^+ and hbox {HCO}_3^- fluxes inside and outside cells. We have validated the model with independent experimental data and used it to investigate how and to which extent tumor cells can survive in adverse micro-environments characterized by acidity and hypoxia. The simulations show a dominance of the hbox {H}^+ exchanges in well-oxygenated regions, and of hbox {HCO}_3^- exchanges in the inner hypoxic regions where tumor cells are known to acquire malignant phenotypes. The model also includes the activity of the enzyme Carbonic Anhydrase 9 (CA9), a known marker of tumor aggressiveness, and the simulations demonstrate that CA9 acts as a nonlinear hbox {pH}_i equalizer at any hbox {O}_2 level in cells that grow in acidic extracellular environments.

Dual activatable self-assembled nanotheranostics for bioimaging and photodynamic therapy

Multifunctional nanosystems that can transport therapeutic and diagnostic agents into tumor sites and activate their respective functions via tumor-microenvironment recognition are highly desirable for clinical applications. We fabricated pH and redox dual-activatable self-assembled nanotheranostics (named as DA-SNs) via coordination-driven self-assembly of chlorin e6 (Ce6) disulfide-linked pH sensitive polymer ligand, poly (isobutylene-alt-maleic anhydride-graft-methoxy-poly (ethyleneglycol)-graft-imidazole-graft-Cystamine-Ce6) [PIMA-mPEG-API-SS-Ce6], and gadolinium ions (Gd3+). DA-SNs exhibited uniform particle size of ~48 nm, excellent stability, and inherent biosafety. Negatively charged DA-SNs could prolong blood circulation time (t1/2 = 2.91 h) and improve tumor accumulation. Moreover, DA-SNs could undergo surface charge switch from negative charge to positive one in a slightly acidic tumor extracellular environment (pH 6.8), thus enhancing cellular uptake. After entering tumor cells, fluorescence, photodynamic therapeutic activity, and T1MR contrast from DA-SNs could be activated within this intracellular environment with lowered pH and high level of GSH. Importantly, human tumors implanted in mice could be successfully visualized via distinct pH and redox dual-sensitive T1MR contrast and fluorescence imaging, indicating that DA-SNs could serve as a dual-modal MR/fluorescence imaging probe for tumor-targeting diagnosis. In addition, DA-SNs exhibited superior photodynamic therapeutic efficiency with negligible side effects. Therefore, this DA-SN shows great promise for synergistic photodynamic therapy and diagnostic imaging.

Phase I dose escalation study of immunoconjugate L-DOS47 in combination with pemetrexed/carboplatin in non-squamous non-small cell lung cancer (NSCLC) patients.

e21680 Background: L-DOS47, a cancer therapeutic designed to exploit the acidic tumour extracellular environment, consists of urease conjugated to an anti-CEACAM6 camelid monoclonal antibody. The antibody serves as a targeting agent to deliver urease to tumor sites, while the urease enzyme converts urea, an abundant natural metabolite, into ammonia and generates a local pH increase. The combined effect of ammonia toxicity and pH increase is cytotoxic to cancer cells. An additive cytotoxic effect of L-DOS47 and pemetrexed/carboplatin has been observed against the A549 human lung adenocarcinoma cell line. L-DOS47 has been shown in a previous phase I clinical trial to be safe and well tolerated at doses up to 13.55 µg/kg. The current phase I study was designed to define the maximum tolerated dose of L-DOS47 in combination with pemetrexed/carboplatin. Methods: Stage IV (TNM M1a and M1b) histologically confirmed non-squamous NSCLC patients (aged ≥18 yrs, ECOG PS ≤1) received up to four cycles of L-DOS47 in combination with standard of care doses of pemetrexed/carboplatin. L-DOS47 was administered weekly over 21 days in each treatment cycle. Seven planned dosing cohorts (0.59, 0.78, 1.5, 3.0, 6.0, 9.0, 12.0 μg/kg) employed a standard 3+3 design for the first two and the last two cohorts, and an accelerated 1 + 2 design for the middle three cohorts. Dose levels of L-DOS47 were escalated following a review of safety data by the Trial Steering Committee. Results: Fourteen (14) patients were enrolled across 6 dosing cohorts before recruitment was halted. Median age was 65.1 years, with 50.0% male and 78.6% Caucasian. 57.1% of patients had an ECOG score of 0, and 42.9% had an ECOG score of 1. The maximum tolerated dose (MTD) could not be determined as none of the patients experienced a dose limiting toxicity (DLT). 50.0% of patients experienced at least one treatment emergent adverse event (TEAE) assessed as study drug-related, with 14.3% of patients experiencing at least one grade 3/4 drug-related toxicity. Of 12 patients evaluable for efficacy, 5 patients (41.7%) had a partial response (PR), 4 patients (33.3%) experienced stable disease (SD) and 3 patients (25.0%) had progressive disease (PD). The objective response rate is 41.7%. The clinical benefit rate is 75.0%. Conclusions: L-DOS47, in combination with pemetrexed/carboplatin, appears to be well tolerated with promising anti-tumor activity against non-squamous NSCLC. Clinical trial information: NCT02309892.

Metal-Organic Framework Nanoparticles Induce Pyroptosis in Cells Controlled by the Extracellular pH.

Ion homeostasis is essential for cellular survival, and elevated concentrations of specific ions are used to start distinct forms of programmed cell death. However, investigating the influence of certain ions on cells in a controlled way has been hampered due to the tight regulation of ion import by cells. Here, it is shown that lipid-coated iron-based metal-organic framework nanoparticles are able to deliver and release high amounts of iron ions into cells. While high concentrations of iron often trigger ferroptosis, here, the released iron induces pyroptosis, a form of cell death involving the immune system. The iron release occurs only in slightly acidic extracellular environments restricting cell death to cells in acidic microenvironments and allowing for external control. The release mechanism is based on endocytosis facilitated by the lipid-coating followed by degradation of the nanoparticle in the lysosome via cysteine-mediated reduction, which is enhanced in slightly acidic extracellular environment. Thus, a new functionality of hybrid nanoparticles is demonstrated, which uses their nanoarchitecture to facilitate controlled ion delivery into cells. Based on the selectivity for acidic microenvironments, the described nanoparticles may also be used for immunotherapy: the nanoparticles may directly affect the primary tumor and the induced pyroptosis activates the immune system.

Stimuli-Responsive Polysaccharide Enveloped Liposome for Targeting and Penetrating Delivery of survivin-shRNA into Breast Tumor.

Silencing the inhibitor of apoptosis (IAP) by RNAi is a promising method for tumor therapy. One of the major challenges lies in how to sequentially overcome the system barriers in the course of the tumor targeting delivery, especially in the tumor accumulation and penetration. Herein we developed a novel stimuli-responsive polysaccharide enveloped liposome carrier, which was constructed by layer-by-layer depositing redox-sensitive amphiphilic chitosan (CS) and hyaluronic acid (HA) onto the liposome and then loading IAP inhibitor survivin-shRNA gene and permeation promoter hyaluronidase (HAase) sequentially. The as-prepared HA/HAase/CS/liposome/shRNA (HCLR) nanocarrier was verified to be stable in blood circulation due to the negative charged HA shield. The tumor targeting recognition and the enhanced tumor accumulation of HCLR were visualized by fluorescence resonance energy transfer (FRET) and in vivo fluorescence biodistribution. The deshielding of HA and the protonizing of CS in slightly acidic tumor extracellular pH environment (pHe, 6.8-6.5) were demonstrated by ζ potential change from -23.1 to 29.9 mV. The pHe-responsive HAase release was confirmed in the tumor extracellular mimicking environments, and the intratumoral biodistribution showed that the tumor penetration of HCLR was improved. The cell uptake of HCLR in pHe environment was significantly enhanced compared with that in physiological pH environment. The increased shRNA release of HCLR was approved in 10 mM glutathione (GSH) and tumor cells. Surprisingly, HCLR suppressed the tumor growth markedly through survivin silencing and meanwhile maintained low toxicity to mice. This study indicates that the novel polysaccharide enveloped HCLR is promising in clinical translation, thanks to the stimuli-triggered tumor accumulation, tumor penetration, cell uptake, and intracellular gene release.

Stepwise targeting and responsive lipid-coated nanoparticles for enhanced tumor cell sensitivity and hepatocellular carcinoma therapy.

Rationale: Antitumor drug delivery faces multiple barriers that require consecutively achieving tumor targeting, selective cellular uptake and sufficient intracellular drug dosage.Methods: Herein, we designed smart nanoparticles (GPDC-MSNs) that can accumulate stepwise in tumor tissues, selectively enter cancer cells by responding to the acidic tumor extracellular environment, and achieve considerable drug release in the intracellular microenvironment. The GPDC-MSNs comprise the synthesized material galactosyl-conjugated PEO-PPO-PEO (Gal-P123) for hepatocellular carcinoma (HCC) targeting, the tumor extracellular pH-responsive lipid (2E)-4-(dioleostearin)-amino-4-carbonyl-2-butenonic (DC) for selective cellular internalization, and antitumor drug irinotecan (CPT-11)-loaded mesoporous silica nanoparticles (MSNs) for on-demand intracellular drug release.Results: GPDC-MSNs are negatively charged at pH 7.4 and promote active HCC targeting mediated by the asialoglycoprotein receptor. Upon reaching the weakly acidic tumor microenvironment, the nanoparticles undergo charge conversion to neutral, enhancing cellular internalization. Moreover, the encapsulated CPT-11 can be retained within GPDC-MSNs in the blood circulation but undergo intracellular burst release, which facilitates the apoptosis of tumor cells. GPDC-MSNs significantly increased HCC selectivity in vivo and exhibited up to 25 times higher accumulation in tumor tissue than in normal hepatic tissue, thus achieving superior antitumor efficacy and low systemic toxicity.Conclusion: This stepwise-responsive nanoparticle should serve as a valuable platform and promising strategy for HCC treatment.

Doped Graphene Quantum Dots for Intracellular Multicolor Imaging and Cancer Detection.

Despite significant advances of nanomedicine, the issues of biocompatibility, accumulation-derived toxicity, and the lack of sensing and in vivo imaging capabilities hamper the translation of most nanocarriers into clinic. To address this, we utilize nitrogen, boron/nitrogen, and sulfur-doped graphene quantum dots (GQDs) as fully biocompatible multifunctional platforms allowing for multicolor visible/near-IR imaging and cancer-sensing. These GQDs are scalably produced in one-step synthesis from a single biocompatible glucosamine precursor, are water-soluble, show no cytotoxicity at high concentrations of 1 mg/mL, and demonstrate substantial degradation at 36 h in biological environments as verified by TEM imaging. Because of their small sizes, GQDs exhibit efficient internalization maximized at 12 h followed by further degradation/excretion. Their high-yield intrinsic fluorescence in blue/green and near-infrared allows for multicolor in vitro imaging on its own or in combination with other fluorophores, and offers the capabilities for in vivo near-IR fluorescence tracking. Additionally, nitrogen- and sulfur-doped GQDs exhibit pH-dependent fluorescence response that is successfully utilized as a sensing mechanism for acidic extracellular environments of cancer cells. It allows for the deterministic, ratiometric spectral discrimination between cancerous (HeLa and MCF-7 cell) versus healthy (HEK-293 cell) environments with substantial intensity ratios of 1.6 to 8. These results suggest fully biocompatible GQDs developed in this work as multifunctional candidates for in vitro delivery of active agents, multicolor visible/near-IR fluorescence imaging, and pH-sensing of cancerous environments.

Stepwise pH-sensitive and biodegradable polypeptide hybrid micelles for enhanced cellular internalization and efficient nuclear drug delivery

The short blood circulation time, reduced cellular uptake, and uncontrollable drug release still hinder the polymer micelle as an efficient drug delivery vehicle in clinical applications. In this study, a series of stepwise pH-sensitive and biodegradable polypeptide hybrid terpolymers, poly (lysine-co-N,N-bis(acryloyl) cystamine-co-dimethylmaleic anhydride) (PLB-DMMA), were designed and synthesized to achieve prolonged circulation time, enhanced cellular uptake and controllable anti-cancer drug release. The synthesized terpolymers can self-assemble into spherical nano-micelles (NMs) with narrow distributions and exhibited stepwise responses to extracellular and intracellular pH condition of the tumor cell. The as prepared NMs showed a negative surface charge under normal physiological conditions exhibiting advantageous stability during blood circulation. By the first-step pH response, the surface charge of the NMs switched from negative to positive to enhance cellular uptake under the slightly acidic tumor extracellular environment. After internalization into tumor cells, the second-step pH response resulted in an endosome escape of the NMs via the “proton-sponge” effect in the acidic endo/lysosome environment. Additionally, a rapid drug release was triggered in response to the intracellular reductive environment of tumor cells via the destruction of disulfide-linked polymer chains to enhance the nucleus delivery of DOX. in vitro cell assays showed that the blank NMs showed negligible systemic toxicity against normal cells while the DOX-loaded NMs significantly inhibited growth of the tumor cells. In general, it was suggested that the as developed stepwise pH-sensitive and biodegradable PLB-DMMA based NMs would be a smart and promising drug delivery candidate for anti-cancer chemotherapy.

PH-Responsive Nano-Self-Assemblies of the Anticancer Drug 2-Hydroxyoleic Acid.

pH-responsive lipid nanocarriers have the potential to selectively target the acidic extracellular pH environment of cancer tissues and may further improve the efficacy of chemotherapeutics by minimizing their toxic side-effects. Here, we present the design and characterization of pH-sensitive nano-self-assemblies of the poorly water-soluble anticancer drug 2-hydroxyoleic acid (2OHOA) with glycerol monooleate (GMO). pH-triggered nanostructural transformations from 2OHOA/GMO nanoparticles with an internal inverse hexagonal structure (hexosomes) at pH around 2.0-3.0, via nanocarriers with an internal inverse bicontinuous cubic structure (cubosomes) at pH 2.0-4.5, to vesicles at pH 4.5-7.4 were observed with synchrotron small-angle X-ray scattering, and cryogenic transmission electron microscopy. ζ-potential measurements highlight that the pH-driven deprotonation of the carboxylic group of 2OHOA, and the resulting charge-repulsions at the lipid-water interface account for these nanostructural alterations. The study provides detailed insight into the pH-dependent self-assembly of 2OHOA with GMO in excess buffer at physiologically relevant pH values, and discusses the effects of pH alterations on modulating their nanostructure. The results may guide the further development of pH-responsive anticancer nanocarriers for the targeted delivery of chemotherapeutics to the local microenvironment of tumor cells.

(Invited) Biocompatible Doped Graphene Quantum Dots Enabling Visible/Near-Infrared Imaging, Drug Delivery and Cancer Detection

Despite significant advances of nanomedicine, the issues of biocompatibility, accumulation-derived toxicity and the lack of sensing and in vivo imaging capabilities hamper the translation of many nanocarriers into clinic. To address these needs we develop and test the feasibility of new nitrogen, sulfur or metal-doped graphene quantum dots (GQDs) as fully biocompatible multifunctional platforms enabling theranostic image-guided approaches both in vitro and in vivo. These GQDs scalably produced in one-step synthesis from a single biocompatible carbon-rich precursor form 3-5 nm few-layer structures functionalized with oxygen-containing groups, however, showing high crystallinity and lattice spacing indicative of a graphitic platform. They are water-soluble, show no cytotoxicity at high concentrations of 1 mg/mL, and demonstrate substantial degradation at 36h in biological environments as verified by TEM imaging. Due to their small sizes, GQDs exhibit efficient internalization maximized at 12 h followed by further degradation/excretion and enable active agent delivery. Their intrinsic fluorescence in blue/green with up to 62% quantum yield and near-infrared allows for multicolor in vitro imaging on its own or in combination with other fluorophores and offers the potential capabilities for in vivo near-IR fluorescence tracking. Additionally, several GQD types exhibit pH-dependent fluorescence response that is successfully utilized as a sensing mechanism for acidic extracellular environments of cancer cells. It allows for deterministic, ratiometric discrimination between cancerous (HeLa and MCF-7 cell) and healthy (HEK-293 cell) environments with substantial spectral peak intensity ratios of 1.6 to 8 in healthy versus cancer cells. Such spectral technique, less prone to quenching discrepancies, provides a useful tool for pH-sensing of microenvironments. These results suggest fully biocompatible doped GQDs as multifunctional candidates for in vitro delivery of active agents, multicolor visible/near-IR fluorescence imaging, and pH-sensing of cancerous environments.

Hierarchical tumor acidity-responsive self-assembled magnetic nanotheranostics for bimodal bioimaging and photodynamic therapy

Nanosized self-assemblies built from inorganic nanoparticles and polymer ligands have the potential to generate personalized theranostics systems for diagnostic imaging and cancer therapy. However, most of the theranostics systems suffer from poor targeting activity, insensitive diagnosis and drug leakage, leading to poor treatment results. In this study, a hierarchical tumor acidity-responsive magnetic nanobomb (termed HTAMN) was developed for photodynamic therapy and diagnostic imaging. The HTAMNs were formed through the self-assembly of chlorin e6 (Ce6)-functionalized polypeptide ligand, methoxy poly (ethyleneglycol)-block-poly (dopamine-ethylenediamine-2,3-dimethylmaleic anhydride)-L-glutamate-Ce6 [mPEG-b-P (Dopa-Ethy-DMMA)LG-Ce6] and superparamagnetic iron oxide nanoparticles (SPIONs). Negatively charged HTAMNs circulate in the blood for prolonged periods and promote tumor retention by passive targeting to the tumor. Once the HTAMNs arrive at the tumor location, the acidic extracellular tumor environment reverses the surface charge of the HTAMNs, resulting in tumor accumulation and cellular uptake. Moreover, in response to the more acidic environment inside cells, the photosensitizers are activated resulted in enhanced diagnostic imaging and cancer treatment. The in vitro and in vivo results indicate the effective tumor accumulation, internalization, diagnostic sensitivity and superior photodynamic therapy effect of the HTAMNs. Therefore, designing smart HTAMNs can promote the rapid development of cancer theranostics for clinical implementation.

The pH Sensitive Atram Peptide Hitchhikes on Human Serum Albumin En Route to Target Diseased Acidic Tissues

Our lab developed the acidity triggered rational membrane (ATRAM) peptide to target the acidic extracellular environment of tumors. Biophysical and cellular experiments have shown that the membrane interaction of ATRAM is pH-dependent. Often rapid enzymatic degradation and renal clearance of peptides in vivo restricts their successful application as a drug delivery system. However, our previous mice studies demonstrated that ATRAM has an extended circulation half-life. We hypothesized that ATRAM might be interacting with a blood component that shields the peptide from degradation. Binding assays confirmed that ATRAM binds to human serum albumin (HSA), a plasma protein, with higher affinity than insulin detemir, a clinically used drug, suggesting that it might bind to this protein in the blood stream. Fluorescence studies established that ATRAM interacts reversibly with HSA, as the peptide partitioned to lipid membranes after pre-incubation with HSA. This result suggests that while that peptide can use albumin as a carrier in the blood stream, it will still target and transfer to the cell membrane. Understanding how ATRAM is able to avoid immediate degradation and renal clearance in vivo can broaden the design and application range of peptides as therapeutics.

Graphene Oxide as a Multifunctional Platform for Intracellular Delivery, Imaging, and Cancer Sensing

Graphene oxide (GO), the most common derivative of graphene, is an exceptional nanomaterial that possesses multiple physical properties critical for biomedical applications. GO exhibits pH-dependent fluorescence emission in the visible/near-infrared, providing a possibility of molecular imaging and pH-sensing. It is also water soluble and has a substantial platform for functionalization, allowing for the delivery of multiple therapeutics. GO physical properties are modified to enhance cellular internalization, producing fluorescent nanoflakes with low (<15%) cytotoxicity at the imaging concentrations of 15 μg/mL. As a result, at lower flake sizes GO rapidly internalizes into HeLa cells with the following 70% fluorescence based clearance at 24 h, assessed by its characteristic emission in red/near-IR. pH-dependence of GO emission is utilized to provide the sensing of acidic extracellular environments of cancer cells. The results demonstrate diminishing green/red (550/630 nm) fluorescence intensity ratios for HeLa and MCF-7 cancer cells in comparison to HEK-293 healthy cells suggesting a potential use of GO as a non-invasive optical sensor for cancer microenvironments. The results of this work demonstrate the potential of GO as a novel multifunctional platform for therapeutic delivery, biological imaging and cancer sensing.

Stepwise dual pH and redox-responsive cross-linked polypeptide nanoparticles for enhanced cellular uptake and effective cancer therapy.

The systemic toxicity, reduced cellular internalization, and uncontrollable intracellular drug release of smart nanoparticles (NPs) still need to be overcome for effective cancer therapy. Herein, a series of stepwise dual pH and redox responsive cross-linked polypeptides based on poly(l-lysine-co-N,N-bis(acryloyl)cystamine-co-γ-glutamic acid) (PLBG), were prepared for enhanced cellular uptake and effective cancer therapy. The prepared cross-linked PLBG nanoparticles (PLBG-NPs) exhibit negatively charged surfaces under physiological conditions, and the surface charge of the PLBG-NPs was observed to switch from negative to positive in a slightly acidic tumor extracellular environment at a pH level of ∼6.5. Finally, the endosome escape of the PLBG-NPs was facilitated via a "proton-sponge" effect via protonation of the polymer chain in the endo/lysosome environment of the tumor cell at a pH level of ∼5.0. Moreover, rapid drug release was triggered by a high concentration of reducing glutathione (GSH) in tumor cells via destruction of the disulfide linkages in the PLBG-NPs. Cytotoxicity assays demonstrated that no noticeable cytotoxicity was observed for the PLBG-NPs. However, DOX-loaded PLBG-NPs (PLBG-NPs-DOX) demonstrated remarkable tumor cell killing effects. In HeLa tumor-bearing mice, PLBG-NPs-DOX showed sustained tumor accumulation and enhanced inhibitory effects on tumor growth and angiogenesis. In conclusion, it is suggested that the as-designed novel charge-conversion PLBG-NPs with stepwise pH-responsivity and biodegradability could be used as a potential drug carrier for effective cancer therapy with less systemic toxicity.

Acidic extracellular environment affects miRNA expression in tumors in vitro and in vivo.

Hypoxia can control the expression of miRNAs in tumors which play an important role for the control of the malignant behavior. The aim of the study was to analyze whether extracellular acidosis, a common feature of tumors, also has an impact on the miRNA expression in isolated cells as well as in solid tumors. MiRNA expression was analyzed in two rat tumor cell lines (AT1 prostate and Walker-256 mammary carcinomas) by NGS and qPCR. In vivo the same cell lines were implanted subcutaneously and the tumor pH was modulated by inspiratory hypoxia and inhibition of the respiratory chain. In addition, the expression of five genes (Brip1, Ercc6l, Ikbke, Per3, Tlr5) which are potential targets of the miRNAs were analyzed on mRNA level. Screening showed that 38 (AT1) resp. 41 (Walker-256) miRNAs were pH-dependent. Validation by qPCR revealed that only 4 miRNAs were consistently regulated in both cell lines: miR-183, miR-203a, miR-215 and miR-7a. The expression of miR-7a was increased by low pH whereas all others were decreased. In the tumors in vivo all 4 miRNAs were down-regulated. The potential targets showed pH dependency in both cell lines. In conclusion, extracellular acidosis regulates the expression of miRNAs in vitro and in vivo. The expression of targets of these miRNAs were also pH-dependent. The pH may therefore affect the biological behavior of tumors via miRNAs.

Interaction of Artepillin C with model membranes: Effects of pH and ionic strength

Artepillin C is the major constituent of green propolis, one of the most consumed products in popular medicine owing to its therapeutic effects, including antitumor activity. Artepillin C differs from other cinnamic acid derivatives due to the presence of two prenylated groups in its structure, believed to enhance access to the cell membrane and resulting in pharmacological activity. The membrane outer leaflet of tumor cells is exposed to an acidic extracellular environment, which could modulate the protonation state of antitumor drugs and hence their interaction with the cell membrane. Herein, we investigated the interaction of Artepillin C with Langmuir monolayers and giant unilamellar vesicles (GUVs) of 1,2‑dipalmitoyl‑sn‑glycerol‑3‑phosphocholine (DPPC) used as model membranes, in physiological and acidic environments. We observed that protonation of the carboxyl group of Artepillin C is essential for the interaction, with larger shifts induced in the surface pressure isotherms of DPPC monolayers in comparison with deprotonated Artepillin C. Also observed was a decrease in lipid packing inferred from the compressibility modulus and Brewster angle microscopy (BAM) images for monolayers on acidic subphases. Results with microscopy techniques on GUVs confirmed that.Artepillin C causes a curvature stress of the lipid bilayer only in its neutral state, causing the GUVs to burst. The stronger effects of neutral Artepillin C on both monolayers and GUVs were maintained when the ionic strength was increased. Taken together, the results indicate that Artepillin C may have preferential attachment to a more acidic environment which might be an important feature for its antitumor activity.

PH-dependent thermodynamic intermediates of pHLIP membrane insertion determined by solid-state NMR spectroscopy

The applications of the pH low insertion peptide (pHLIP) in cancer diagnosis and cross-membrane cargo delivery have drawn increasing attention in the past decade. With its origin as the transmembrane (TM) helix C of bacteriorhodopsin, pHLIP is also an important model for understanding how pH can affect the folding and topogenesis of a TM α-helix. Protonations of multiple D/E residues transform pHLIP from an unstructured coil at membrane surface (known as state II, at pH ≥ 7) to a TM α-helix (state III, pH ≤ 5.3). While these initial and end states of pHLIP insertion have been firmly established, what happens at the intervening pH values is less clear. However, the intervening pH range is most relevant to pHLIP-cell interactions in the acidic extracellular tumor environment (and in the endosomes within cells). Here, using advanced solid-state NMR spectroscopy with palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine unilamellar vesicles as the model membrane, we systematically examined the state of pHLIP-membrane interactions (in terms of the membrane locations of D/E residues, as well as lipid dynamics) at the intervening pH values of 6.4, 6.1, and 5.8, along with the known states at pH 7.4 and 5.3. Thermodynamic intermediate states distinct from the initial and end states were discovered to exist at each of the intervening pH examined. They support a multistage model of pHLIP insertion in which the D/E titrations occur in a defined sequence at distinct intermediate pH values. This multistage model has important ramifications in pHLIP applications.

In Situ Imaging of Live-Cell Extracellular pH during Cell Apoptosis with Surface-Enhanced Raman Spectroscopy

Extracellular pH (pHe) is an important regulating factor that determines many cellular processes, including proliferation, differentiation, and apoptosis. In our previous work, we developed 4-MPy (4-mercaptopyridine) modified Au nanoparticles as intracellular pH sensors based on surface-enhanced Raman spectroscopy (SERS). We herein modified a Au-nanoparticle-assembled solid SERS substrate with 4-MPy molecules for in situ pHe sensing during apoptosis. We found a more acidic extracellular environment of cancer cells than that of normal cells from the pH imaging. We then in situ investigated the temporal and spatial evolution of pHe of cancer cells after addition of transforming growth factor-β (TGF-β). The pHe showed a fast decrease at the beginning, followed by a slow decrease until the complete loss of cellular functions, and the pH values in and out of the cells became similar. This work shows that our SERS substrate combined with an in situ cell culture system is well suitable for in situ pHe sensing during cell processes and will be a promising technique for understanding more pHe-related biological and pathological issues.