Intracellular Microenvironment Research Articles

Absolutely "off-on" fluorescent CD-based nanotheranostics for tumor intracellular real-time imaging and pH-triggered DOX delivery.

Carbon dots (CDs) have attracted intense attention in tumor nanotheranostics recently; however, those nanotheranostics exhibited similar fluorescence in both normal and tumor tissues, limiting their practical application. In the present work, absolutely "off-on" fluorescent CD-based nanotheranostics was designed for tumor intracellular real-time imaging and pH-triggered DOX delivery via both static quenching by the crosslinking of benzaldehyde-containing diblock copolymers and dynamic quenching because of the surrounding conjugated DOX molecules. The proposed PPEGMA42-b-PFPMA122-(CDs)-DOX nanotheranostics did not exhibit fluorescence in a normal physiological medium, while strong fluorescence recovery occurred in the tumor intracellular microenvironment due to pH-triggered disintegration, releasing the CDs and DOX. The pH-triggered DOX release and absolute "off-on" fluorescence make the proposed nanotheranostics promising for tumor-specific pH-triggered DOX delivery and imaging.

Facile Fabrication of Redox-Responsive Covalent Organic Framework Nanocarriers for Efficiently Loading and Delivering Doxorubicin.

Covalent organic frameworks (COFs) as drug delivery systems have shown great promise, but their pharmaceutical applications are often limited by complex building blocks, tedious preparations, irregular shape, and uncontrolled drug release within target cells. Herein, a facile strategy is developed to prepare PEGylated redox-responsive nanoscale COFs (denoted F68@SS-COFs) for efficiently loading and delivering doxorubicin (DOX) by use of FDA-approved Pluronic F68 and commercially available building blocks. The obtained F68@SS-COFs with controlled size, high stability, and good biocompatibility can not only achieve a very high DOX-loading content (about 21%) and very low premature leakage at physiological condition but can also rapidly respond to the tumor intracellular microenvironment and efficiently release DOX to kill tumor cells. Considering the readily available raw materials, simple preparation process, and desirable redox-responsiveness, the strategy provided here opens up a promising avenue to develop well-defined COFs-based nanomedicines for cancer therapy.

Self-responsive co-delivery system for remodeling tumor intracellular microenvironment to promote PTEN-mediated anti-tumor therapy.

Delivering the pten gene into tumor cells to reacquire PTEN functionality is considered to be an attractive method for cancer treatment. However, the inhibition effect of the tumor intracellular microenvironment (TIME), especially at the high reactive oxygen species (ROS) level, on pten expression and PTEN protein functionality was nearly overlooked. Herein, the development of a potential strategy is described, which enhances PTEN-mediated anti-tumor capability by exhausting the intracellular ROS in TIME. To achieve this, poly(ethyleneimine) (PEI)-modified DSPE was introduced to protect the pten plasmid, and form liposomes for encapsulating the "scavenger" of oxidation homeostasis, epigallocatechin-3-gallate (EGCG). Notably, this was a simple system with improved safety compared which when compared with the use of PEI could accomplish efficient pten transfection and simultaneous disintegration to cause transient release of EGCG responding to the endosome environment through the "proton sponge effect". In the cytoplasm, EGCG depleted ROS and promoted the expression of the pten gene as well as restoring protein functionality, thus negatively regulating the PI3K-AKT signaling pathway. In vitro and in vivo results revealed that this system significantly inhibited tumor growth via remodeling of the TIME, and provided a promising way to control malignant tumors.

Landscape of gene expression variation of natural isolates of Cryptococcus neoformans in response to biologically relevant stresses.

Cryptococcus neoformans is an opportunistic fungal pathogen that at its peak epidemic levels caused an estimated million cases of cryptococcal meningitis per year worldwide. This species can grow in diverse environmental (trees, soil and bird excreta) and host niches (intracellular microenvironments of phagocytes and free-living in host tissues). The genetic basic for adaptation to these different conditions is not well characterized, as most experimental work has relied on a single reference strain of C. neoformans. To identify genes important for yeast infection and disease progression, we profiled the gene expression of seven C. neoformans isolates grown in five representative in vitro environmental and in vivo conditions. We characterized gene expression differences using RNA-Seq (RNA sequencing), comparing clinical and environmental isolates from two of the major lineages of this species, VNI and VNBI. These comparisons highlighted genes showing lineage-specific expression that are enriched in subtelomeric regions and in lineage-specific gene clusters. By contrast, we find few expression differences between clinical and environmental isolates from the same lineage. Gene expression specific to in vivo stages reflects available nutrients and stresses, with an increase in fungal metabolism within macrophages, and an induction of ribosomal and heat-shock gene expression within the subarachnoid space. This study provides the widest view to date of the transcriptome variation of C. neoformans across natural isolates, and provides insights into genes important for in vitro and in vivo growth stages.

Targeted pH-responsive polyion complex micelle for controlled intracellular drug delivery

Cancer therapy with nanoscale drug formulations has made significant progress in the past few decades. However, the selective accumulation and release of therapeutic agents in the lesion sites are still great challenges. To this end, we developed a cRGD-decorated pH-responsive polyion complex (PIC) micelle for intracellular targeted delivery of doxorubicin (DOX) to upregulate tumor inhibition and reduce toxicity. The PIC micelle was self-assembled via the electrostatic interaction between the positively charged cRGD-modified poly(ethylene glycol)-block-poly(l-lysine) and the anionic acid-sensitive 2,3-dimethylmaleic anhydride-modified doxorubicin (DAD). The decoration of cRGD enhanced the cell internalization of PIC micelle through the specific recognition of αvβ3 integrin on the membrane of tumor cells. The active DOX was released under intracellular acidic microenvironment after endocytosis following the decomposition of DAD. Moreover, the targeted PIC micelle exhibited enhanced inhibition efficacies toward hepatoma in vitro and in vivo compared with the insensitive controls. The smart multifunctional micelle provides a promising platform for target intracellular delivery of therapeutic agent in cancer therapy.

Quercetin@ZIF-90 as a novel antioxidant for label-free colorimetric ATP sensing at neutral pH

Quercetin is a nature polyphenolic flavonoid compound, which act as antioxidant and radical scavengers in cells. However, such activities strongly depend on the intracellular microenvironment, which has become an urgency to develop a more stable method for better use of quercetin. Herein, we report a facile approach to fabricate Quercetin@ZIF-90 (QZ) with a narrow size distribution about 100 nm, the synthesized QZ exhibits considerable antioxidant and radical scavenger ability comparing to bare quercetin. Moreover, the experiment results indicated that QZ was sensitive to adenosine 5′-triphosphate (ATP) and pH, thus QZ was applied as a novel antioxidant for the determination of ATP. In brief, though 3, 3′, 5, 5′-tetramethylbenzidine (TMB) could be oxidized to blue cation radical complex by oxidase-like BSA-MnO2 nanosheets (MNs) at neutral pH, QZ could sufficiently protect against MNs-induced TMB cation radicals reaction, which means no obvious color change in MNs-TMB system after the addition of QZ. However, ATP could induced collapse of QZ due to the interaction between ATP and Zn2+ allowing the release of quercetin from ZIF-90 shell, and the inhibition effect to the MNs was weakened, leading to the recovery of blue oxTMB. In this way, a novel “Off-On” colorimetric method for ATP sensing was established, and the method displayed a good linear relationship ranging from 2.0 μ mol L−1 to 80.0 μ mol L−1. The reported strategy is also capable of ATP sensing in MCF-7 cell lysates, which illustrated our method exhibited high sensitivity and selectivity.

Multifunctional Branched Nanostraw-Electroporation Platform for Intracellular Regulation and Monitoring of Circulating Tumor Cells.

Downstream analysis of circulating tumor cells (CTCs) has provided new insights into cancer research. In particular, the detection of CTCs, followed by the regulation and monitoring of their intracellular activities, can provide valuable information for comprehensively understanding cancer pathogenesis and progression. However, current CTC detection techniques are rarely capable of in situ regulation and monitoring of the intracellular microenvironments of cancer cells over time. Here, we developed a multifunctional branched nanostraw (BNS)-electroporation platform that could effectively capture CTCs and allow for downstream regulation and monitoring of their intracellular activities in a real-time and in situ manner. The BNSs possessed numerous nanobranches on the outer sidewall of hollow nanotubes, which could be conjugated with specific antibodies to facilitate the effective capture of CTCs. Nanoelectroporation could be applied through the BNSs to nondestructively porate the membranes of the captured cells at a low voltage, allowing the delivery of exogenous biomolecules into the cytosol and the extraction of cytosolic contents through the BNSs without affecting cell viability. The efficient delivery of biomolecules (e.g., small molecule dyes and DNA plasmids) into cancer cells with spatial and temporal control and, conversely, the repeated extraction of intracellular enzymes (e.g., caspase-3) for real-time monitoring were both demonstrated. This technology can provide new opportunities for the comprehensive understanding of cancer cell functions that will facilitate cancer diagnosis and treatment.

Boron mitigates citrus root injuries by regulating intracellular pH and reactive oxygen species to resist H+-toxicity

Boron (B)-deficiency and H+-toxicity are important limiting factors for plants growth in acid soils. High B supply may reduce H+-toxicity-induced inhibition of growth in citrus. Trifoliate orange rootstock seedlings were irrigated with nutrient solution containing either 0 μM or 10 μM H3BO3 at two pH levels (pH4 (H+-toxicity) and pH6 (normal)). The results showed that H+-toxicity without B severely hampered main root elongation. Simultaneously, oxidative damage caused by H+-toxicity led to severe damage to the apical structure of root such as root crown abscission. However, B application promoted the root length, root cell viability and reduced cell wall (CW) thickness of root tips under H+-toxicity. Additionally, B application reduced the H+-toxicity-induced reactive oxygen species (ROS) accumulation in roots as characterized by lower fluorescence intensity of H2O2 and O2- staining. Moreover, 31P-NMR (31P nuclear magnetic resonance) spectra revealed B application regulated the pH of vacuoles and cytoplasm in root tips by reducing phosphoenolpyruvate carboxykinase (PEPCase) activity while enhancing NADP malic enzyme (NADP-ME) activity during H+-toxicity. Collectively, our results demonstrate that B supply alleviates H+-toxicity and promotes root growth by reducing ROS accumulation, attenuating intracellular acidic microenvironment to ensure normal chemical reactions in root tip cells.

Aminonaphthalimide hybrids of mitoxantrone and amonafide as anticancer and fluorescent cellular imaging agents

Novel water-soluble 4-aminonaphthalimides were synthesised and their cellular fluorescent imaging, cytotoxicity and ability to induced apoptosis evaluated. The lead compound 1 was designed from the cross-fertilisation of the basic hydrophilic amino pharmacophore of mitoxantrone, and an aminonaphthalimide scaffold of the drug candidate, amonafide. The compounds are also fluorescent pH probes based on photoinduced electron transfer (PET) and internal charge transfer (ICT). The compounds are sensitive to solvent polarity with large Stoke shifts (>90 nm) and provide emissive-coloured solutions (blue to yellow). Excited state pKas of 9.0–9.3 and fluorescence quantum yields of 0.47–0.58 were determined in water. The cytotoxicity and cellular fluorescent imaging properties of the compounds were tested on human cancer cell lines K562 and MCF-7 by the MTT assay, phase contrast and fluorescence microscopy. Compounds 1 and 3 with flexible aminoalkyl chains exhibited GI50 comparable to amonafide, while 2 and 4 with a rigid piperazine moiety and butyl chain are less cytotoxic. Fluorescence microscopy with 1 allowed for the visualization of the intracellular microenvironment exemplifying the potential utility of such hybrid molecules as anticancer and fluorescent cellular imaging agents.

Implementation and application of FRET–FLIM technology

With the development of the new detection methods and the function of fluorescent molecule, researchers hope to further explore the internal mechanisms of organisms, monitor changes in the intracellular microenvironment, and dynamic processes of molecular interactions in cells. Fluorescence resonance energy transfer (FRET) describes the energy transfer process between donor fluorescent molecules and acceptor fluorescent molecules. It is an important means to detect protein–protein interactions and protein conformation changes in cells. Fluorescence lifetime imaging microscopy (FLIM) enables noninvasive measurement of the fluorescence lifetime of fluorescent particles in vivo. The FRET–FLIM technology, which is use FLIM to quantify and analyze FRET, enables real-time monitoring of dynamic changes of proteins in biological cells and analysis of protein interaction mechanisms. The distance between donor and acceptor and their respective fluorescent lifetime, which are of great importance for studying the mechanism of intracellular activity can be obtained by data analysis and algorithm fitting.

Reduction-responsive polypeptide nanomedicines significantly inhibit progression of orthotopic osteosarcoma

Osteosarcoma (OS) is the most common malignant bone tumor with high metastasis and mortality. Neoadjuvant chemotherapy is an effective therapeutic regimen, but the clinical application is limited by the unsatisfactory efficacies and considerable side effects. In this study, the reduction-responsive polypeptide micelles based on methoxy poly(ethylene glycol)-block-poly(S-tert-butylmercapto-L-cysteine) copolymers (mPEG113-b-PBMLC4, P4M, and mPEG113-b-PBMLC9, P9M) were developed to control the delivery of doxorubicin (DOX) in OS therapy. Compared to free DOX, P4M/DOX and P9M/DOX exhibited 2.6 and 3.5 times increase in the area under the curve of pharmacokinetics, 1.6 and 2.0 times increase in tumor accumulation, and 1.6 and 1.7 times decrease of the distribution in the heart. Moreover, the selective accumulation of micelles, especially P9M/DOX, in tumors induced stronger antitumor effects on both primary and lung metastatic OSs with less systematic toxicity. These micelles with smart responsiveness to intracellular microenvironments are highly promising for the targeted delivery of clinical chemotherapeutic drugs in cancer therapy.

HPMA Polymeric Nanocarriers for Anticancer Drugs with Tumor Microenvironment-Responsive Extracellular Biodegradation and Intracellular Drug Release.

The balance between systemic toxicity and circulation time for a polymeric nanocarrier to deliver antitumor drugs has been trialed. A new approach to break the balance was proposed in this study by significantly improving its biosafety and prolonging the circulation time, hence, to enhance its anti-tumor efficacy. A matrix metalloproteinases (MMPs)-sensitive peptide (PVGLIGK) was introduced to cross-link the N-(2-hydroxypropyl) methylacrylamide polymer-doxorubicin conjugates (HPMA-Dox) conjugate to construct a nano-size polymeric nanocarrier-Dox assembly (PMD) with a molecular weight (MW) of 73 kDa and this modification has resulted in a prolonged circulation time (a half-time of 20.1 h) and enhanced accumulation of PMD at the tumor site, while negligible systemic toxicity and excellent biocompatibility were displayed after injection of PMD into the mice. The cross-linked nanoassembly was unpacking in the presence of MMPs in the extracellular microenvironment, and the conjugated Dox was released from the nanoassembly in the lysosome/endosome due to an intracellular low pH microenvironment. The released Dox from PMD inhibited tumor cells very efficiently with a tumor growth inhibition of around 70%. The outstanding performance of the dual stimuli-responsive biodegradable polymeric nanocarriers may open a door for other hydrophobic anti-tumor drugs.

Probing the impact of sulfur/selenium/carbon linkages on prodrug nanoassemblies for cancer therapy

Tumor cells are characterized as redox-heterogeneous intracellular microenvironment due to the simultaneous overproduction of reactive oxygen species and glutathione. Rational design of redox-responsive drug delivery systems is a promising prospect for efficient cancer therapy. Herein, six paclitaxel-citronellol conjugates are synthesized using either thioether bond, disulfide bond, selenoether bond, diselenide bond, carbon bond or carbon-carbon bond as linkages. These prodrugs can self-assemble into uniform nanoparticles with ultrahigh drug-loading capacity. Interestingly, sulfur/selenium/carbon bonds significantly affect the efficiency of prodrug nanoassemblies. The bond angles/dihedral angles impact the self-assembly, stability and pharmacokinetics. The redox-responsivity of sulfur/selenium/carbon bonds has remarkable influence on drug release and cytotoxicity. Moreover, selenoether/diselenide bond possess unique ability to produce reactive oxygen species, which further improve the cytotoxicity of these prodrugs. Our findings give deep insight into the impact of chemical linkages on prodrug nanoassemblies and provide strategies to the rational design of redox-responsive drug delivery systems for cancer therapy.

Click-Type Protein-DNA Conjugation for Mn2+ Imaging in Living Cells.

A click-type protein-DNA conjugation, named as MnDDC (Mn2+-activated DCV-DNA conjunction), is presented, where DCV (rep protein of duck circovirus) and its target DNA work as the modular blocks to rapidly and effectively generate Mn2+-dependent and site-specific protein-DNA linkage. On the basis of MnDCC, a fluorescent Mn2+ biosensor composed of DCV and a molecular beacon, was developed for rapid sensing of Mn2+ within 2 min with nanomolar sensitivity. Using the proposed biosensor, not only analysis of Mn2+ in real samples (e.g., serum and food), but also wash-free fluorescent imaging of Mn2+ in extracellular environment and cytoplasm have been achieved. Moreover, the MnDDC-based sensor was proved to be a powerful tool for visualization of Mn2+ during exploration of the associated cytotoxicity in living neural cells, which is helpful to reveal the cellular responses toward the disordered homeostasis of Mn2+ in both extracellular and intracellular microenvironments.

Branched DNA Architectures Produced by PCR-Based Assembly as Gene Compartments for Cell-Free Gene-Expression Reactions.

The physical distance between genes plays important roles in controlling gene expression reactions in vivo. Herein, we report the design and synthesis of a branched gene architecture in which three transcription units are integrated into one framework through assembly based on the polymerase chain reaction (PCR), together with the exploitation of these constructs as "gene compartments" for cell-free gene expression reactions, probing the impact of this physical environment on gene transcription and translation. We find that the branched gene system enhances gene expression yields, in particular at low concentrations of DNA and RNA polymerase (RNAP); furthermore, in a crowded microenvironment that mimics the intracellular microenvironment, gene expression from branched genes maintains a relatively high level. We propose that the branched gene assembly forms a membrane-free gene compartment that resembles the nucleoid of prokaryotes and enables RNAP to shuttle more efficiently between neighboring transcription units, thus enhancing gene expression efficiency. Our branched DNA architecture provides a valuable platform for studying the influence of "cellular" physical environments on biochemical reactions in simplified cell-free systems.

Polymer microspheres with high drug-loading capacity via dual-modal drug-loading for modulating controlled release property in pH/reduction dual-responsive tumor-specific intracellular triggered doxorubicin release

pH/Reduction dual-responsive poly(4-formylphenyl acrylate-co-acryloyl-β-cyclodextrin) (P(FPA-co-ACD)) microspheres were synthesized as multi-functional vehicle for tumor-specific intracellular triggered doxorubicin (DOX) release, via emulsion copolymerization of acryloyl-β-cyclodextrin (ACD) and 4-formylphenyl acrylate (FPA), with N,N-bis(acryloyl)cystamine (BACy) as crosslinker. In comparison with the drug delivery system (DDS) via mono-modal drug-loading, the DOX/P(FPA-co-ACD)C via only covalent conjugation, or the DOX/P(FPA-co-ACD)N via only host-guest inclusion, the dual-modal drug-loaded DDS, DOX/P(FPA-co-ACD)C+N, were fabricated with a higher drug-loading capacity (DLC) of >53% via both acid-labile Schiff-base conjugation and host-guest inclusion complexation. Furthermore, the DOX release rate from the DOX/P(FPA-co-ACD)C+N was faster than the DOX/P(FPA-co-ACD)C but slower than the DOX/P(FPA-co-ACD)N in the simulated tumor intracellular microenvironment, demonstrating the controlled release behavior of such DDS could be modulated with dual-modal drug-loading.

One-Pot Synthesis of Chicken-Feather-Keratin-Based Prodrug Nanoparticles with High Drug Content for Tumor Intracellular DOX Delivery.

pH/reduction dual-triggered chicken-feather-keratin-based prodrug nanoparticles (C-PK/- SS-Hy-D NPs) were designed via a facile one-pot oxidation coupling reaction between the thiol-functional acid-labile prodrug M-Hy-D and the PEGylated keratin (PK) graft copolymer, for tumor intracellular doxorubicin (DOX) delivery. Due to the encapsulation of the pH and the reduction of the dual-responsive small prodrug D-Hy- SS-Hy-D, a high drug content of 45.8% was obtained in the proposed prodrug nanoparticles. They exhibited excellent pH and reduction of dual-triggered drug release, with cumulative drug release of 88.6% within 51 h in the simulated tumor intracellular microenvironment, while the premature drug leakage was only 13.7% in the simulated normal physiological medium. The in vitro experiments revealed the enhanced antitumor efficacy of the C-PK/- SS-Hy-D NPs than the free DOX at a higher dosage of >10 μg/mL.

Elevated O-GlcNAcylation enhances pro-inflammatory Th17 function by altering the intracellular lipid microenvironment

Chronic, low-grade inflammation increases the risk for atherosclerosis, cancer, and autoimmunity in diseases such as obesity and diabetes. Levels of CD4+ T helper 17 (Th17) cells, which secrete interleukin 17A (IL-17A), are increased in obesity and contribute to the inflammatory milieu; however, the relationship between signaling events triggered by excess nutrient levels and IL-17A-mediated inflammation is unclear. Here, using cytokine, quantitative real-time PCR, immunoprecipitation, and ChIP assays, along with lipidomics and MS-based approaches, we show that increased levels of the nutrient-responsive, post-translational protein modification, O-GlcNAc, are present in naive CD4+ T cells from a diet-induced obesity murine model and that elevated O-GlcNAc levels increase IL-17A production. We also found that increased binding of the Th17 master transcription factor RAR-related orphan receptor γ t variant (RORγt) at the IL-17 gene promoter and enhancer, as well as significant alterations in the intracellular lipid microenvironment, elevates the production of ligands capable of increasing RORγt transcriptional activity. Importantly, the rate-limiting enzyme of fatty acid biosynthesis, acetyl-CoA carboxylase 1 (ACC1), is O-GlcNAcylated and necessary for production of these RORγt-activating ligands. Our results suggest that increased O-GlcNAcylation of cellular proteins may be a potential link between excess nutrient levels and pathological inflammation.

A solvatofluorochromic silicon-substituted xanthene dye useful in bioimaging

In this work, we have performed an in-depth study of the photophysics and solvatofluorochromism of a red-emitting Si-xanthene dye, an analog of Tokyo Magenta (TM) historically developed by Egawa et al. (Chem. Commun.2011, 47, 4162–4164). The results show a strong dependency of the emission properties of 2-Me-4-OMe-TM on the polarity of the solvent. For instance, the dye exhibited an increase in its fluorescence lifetime with the decrease in solvent polarity. Therefore, in this work, this spectral behavior has been used as a new approach for determining the intracellular microenvironment polarity through the measurement of its fluorescence lifetime by Fluorescence Lifetime Imaging Microscopy (FLIM). Our experiments confirmed the ability of the dye to detect changes in polarity between different intracellular compartments.

Capping Silica Nanoparticles with Tryptophan-Mediated Cucurbit[8]uril Complex for Targeted Intracellular Drug Delivery Triggered by Tumor-Overexpressed IDO1 Enzyme.

Nanosystems responsive to tumor-specific enzymes are considered as a highly attractive approach to intracellular drug release for targeted cancer therapy. Mesoporous silica nanoparticles are capped with tryptophan-mediated cucurbit[8]uril complex with Fe3 O4 to minimize the premature drug leakage while being able to deliver the payload on demand at the target tissue. The supramolecular interaction between tryptophan and cucurbit[8]uril is disrupted in the presence of indoleamine 2,3-dioxygenase 1 (IDO1) enzyme (abundant in the tumor intracellular microenvironment), which catalyzes the metabolism of tryptophan into N-formylkynurenine, resulting in the disassembly of the "gate-keeper" of the nanocarriers and intracellular release of therapeutics exclusively in tumor cells. The drug release from the nanocarrier with high selectivity to overexpressed IDO1 enzyme induces significant cytotoxicity against HepG2 cells in vitro, as well as the superior antitumor effects in vivo. This robust supramolecular nanosystem with sophisticated structure and property provides a promising platform for intracellular drug release targeting the intrinsic microenvironmental enzyme inside the tumor cells.