Noninvasive Early Detection of Calpain 2-Enriched Non-Small Cell Lung Cancer Using a Human Serum Albumin-Bounded Calpain 2 Nanosensor.

Lung cancer is diagnosed at an advanced stage due to its unrecognized symptoms, resulting in high mortality. In recent decades, research into the development of an early diagnostic method for lung cancer has expanded in order to overcome the high mortality rate. Calpain 2 (CAPN2) has been suggested as a tumor marker linked to angiogenesis, cell proliferation, and migration in non-small cell lung cancer. In this study, CAPN2 enzyme-activatable near-infrared peptide sensor linked to human serum albumin (HSA-CAPN2) was developed. Intracellular localization and strong recovered fluorescence signals of HSA-CAPN2 were observed in in vitro experiments using A549-Luc cells, and signal recovery was inhibited by ALLN (a CAPN2 inhibitor). In vivo distribution and signal recovery evaluations performed using A549-Luc cell xenograft mice revealed that HSA-CAPN2 accumulated in the tumor region and produced high fluorescent signal recovery. Three-dimensional reconstructed images using single-plane illumination microscopy after tissue clarity visualized localization of HSA-CAPN2 in tumors. In addition, ALLN pretreatment showed a significant inhibitory effect on signal recovery of HSA-CAPN2, and that inhibition was induced by downregulation of CAPN2 at the gene and protein levels followed by decreases in Ca2+ levels. Overall, the results demonstrate the potential of HSA-CAPN2 as a sensor for CAPN2-enriched cancer.

Cellular uptake efficiency of nanoparticles investigated by three-dimensional imaging.

Understanding the interaction of nanoparticles with living cells on the basis of cellular uptake efficiency is a fundamental requisite in biomedical research. Cellular internalization of nanoparticles takes place by mechanisms like ATP hydrolysis-driven endocytosis that deliver nanoparticles to the cytoplasm, organelles and nuclei. Despite its importance in nanomedicine, this uptake procedure is not understood in-depth because of the complexity of the biochemical mechanisms and the lack of available experimental methods for quantitative analysis. The only breakthrough is likely to be the development of imaging techniques that can visualize, monitor and even count the number of nanoparticles inside the cell. To this end, we report here a new, fast and background-free three-dimensional (3-D) imaging technique with quantitative evaluation of the uptake efficiency for NaYF4:Yb3+,Er3+/NaYF4 core/shell upconversion nanoparticles (UCNPs) functionalized with different chemical and biological groups. Furthermore, the multiple 3-D trajectories of the UCNPs have been analyzed to investigate the cellular dynamics. This study reveals the nuclear uptake of UCNPs to be dependent on the specific chemical groups conjugated to the UCNPs. The developed 3-D imaging technique is of great significance for exploring complex biological systems.

Distinct mechanisms for the upconversion of NaYF4:Yb3+,Er3+ nanoparticles revealed by stimulated emission depletion.

Upconversion nanoparticles (UCNPs) have attracted enormous interest over the past few years because of their unique optical properties and potential for use in various applications such as bioimaging probes, biosensors, and light-harvesting materials for photovoltaics. The improvement of imaging resolution is one of the most important goals for UCNPs used in biological applications. Super-resolution imaging techniques that overcome the fundamental diffraction limit of light rely on the photochemistry of organic dyes or fluorescent proteins. Here we report our progress toward super-resolution microscopy with UCNPs. We found that the red emission (655 nm) of core/shell UCNPs with the structure NaYF4:Yb3+,Er3+/NaYF4 could be modulated by emission depletion (ED) of the intermediate state that interacts resonantly with an infrared beam (1540 nm). In contrast, the green emission bands (525 and 545 nm) of the UCNPs were less affected by irradiation with the infrared beam. The origin of such distinct behaviors between the green and red emissions was attributed to their different photophysical pathways.

Small-molecule inhibitors of USP7 induce apoptosis through oxidative and endoplasmic reticulum stress in cancer cells.

USP7 is a deubiquitinating enzyme that involves the ubiquitin proteasome system (UPS) to maintain regulation of protein synthesis and degradation. The well-known substrate of USP7 is the Mdm2-p53 complex. In fact, several studies have reported that functional inhibition of USP7 induces cancer cell apoptosis through activation of p53. However, the contribution of oxidative or endoplasmic reticulum (ER) stress, which is commonly induced by inhibition of the UPS for USP7 inhibitor-mediated apoptosis in cancer cells, has not been investigated. In contrast to previous reports, we show that p53 is not critical during USP7 inhibitor-induced apoptosis in several cancer cells. Inhibition of deubiquitinating enzyme activities by USP7 inhibitors causes ER stress by accumulating polyubiquitinated proteins in cancer cells. Furthermore, we demonstrate that USP7 inhibitors increase intracellular reactive oxygen species and mainly cause cancer cell apoptosis. Taken together, our results reveal that oxidative and ER stress, rather than the Mdm2-p53 axis, mainly contributes to USP7 inhibitor-mediated apoptosis in cancer cells.

PRMT5 is essential for the eIF4E-mediated 5'-cap dependent translation.

It is becoming clear that PRMT5 plays essential roles in cell cycle progression, survival, and responses to external stresses. However, the precise mechanisms underlying such roles of PRMT5 have not been clearly understood. Previously, we have demonstrated that PRMT5 participates in cellular adaptation to hypoxia by ensuring 5'-cap dependent translation of HIF-1α. Given that c-Myc and cyclin D1 expressions are also tightly regulated in 5'-cap dependent manner, we here tested the possibility that PRMT5 promotes cell proliferation by increasing de novo syntheses of the oncoproteins. c-Myc and cyclin D1 were found to be noticeably downregulated by PRMT5 knock-down. A RNA immunoprecipitation analysis, which can identify RNA-protein interactions, showed that PRMT5 is required for the interaction among eIF4E and 5'-UTRs of HIF-1α, c-Myc and cyclin D1 mRNAs. In addition, PRMT5 knock-down inhibited cell proliferation by inducing cell cycle arrest at the G1 phase. More importantly, ectopic expression of eIF4E significantly rescued the cell cycle progression and cell proliferation even in PRMT5-deficeint condition. Based on these results, we propose that PRMT5 determines cell fate by regulating 5'-cap dependent translation of proteins essential for proliferation and survival.

Highly Air-Stable, Flexible, and Water-Resistive 2D Titanium Carbide MXene-Based RGB Organic Light-Emitting Diode Displays for Transparent Free-Form Electronics.

Flexible see-through displays are considered to be the next generation smart display, providing improved information flow, safety, situational awareness, and overall user experience in smart windows, automotive displays, glass-form biomedical displays, and augmented reality systems. 2D titanium carbides (MXenes) are promising material as electrodes of the transparent and flexible displays due to their high transparency, metallic conductivity, and flexibility. However, current MXene-based devices have insufficient air stability and lack engineering schemes to develop matrix-addressable display forms with sufficient pixels to display information. Here, we develop an ultraflexible and environmentally stable MXene-based organic light-emitting diode (OLED) display by combining high performance MXene electrodes, flexible OLEDs, and ultrathin and functional encapsulation systems. The MXene material was synthesized and used to fabricate a highly reliable MXene-based OLED that can stably operate in air condition for over 2000 h, endure repetitive bending deformation of 1.5 mm radius, and maintain environmental stability for 6 h when exposed to wet surroundings. The RGB MXene-based OLEDs were fabricated, (1691 cd m-2 at 40.4 mA cm-2 for red, 1377 cd m-2 at 4.26 mA cm-2 for green, and 1475 cd m-2 at 18.6 mA cm-2 for blue) and a matrix-addressable transparent OLED display was demonstrated that could display letters and shapes.

pH-sensitive multi-drug liposomes targeting folate receptor ß for efficient treatment of non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the leading cause of lung cancer-related deaths worldwide. Tumor-associated macrophages (TAMs), which can be polarized into tumor-promoting M2 phenotype, overexpress folate receptor beta (FRß) and are associated with poor prognosis in NSCLC. In addition, calpain-2 (CAPN2) is overexpressed in NSCLC and is involved in tumor growth. To improve the anticancer efficacy of drugs and reduce their side effects in the treatment of NSCLC, it is important to develop smart drug delivery systems with specific targeting ability and controlled release mechanisms. In this study, FRß-targeted pH-sensitive liposomes were designed as carriers to ensure efficient drug delivery and acid-responsive release in NSCLC cells. Folate-mediated targeting of FRß in M2 TAMs and NSCLC cells effectively inhibited tumor growth and the stimulus-responsive drug release reduced the toxic side effects of the drug. The combination of doxycycline (anti-CAPN2) and docetaxel (anticancer drug) showed a synergistic inhibitory effect on tumor growth by suppressing CAPN2 expression.

Platelet-Like Gold Nanostars for Cancer Therapy: The Ability to Treat Cancer and Evade Immune Reactions.

The cell membrane-coating strategy has opened new opportunities for the development of biomimetic and multifunctional drug delivery platforms. Recently, a variety of gold nanoparticles, which can combine with blood cell membranes, have been shown to provide an effective approach for cancer therapy. Meanwhile, this class of hybrid nanostructures can deceive the immunological system to exhibit synergistic therapeutic effects. Here, we synthesized red blood cell (RBC) and platelet membrane-coated gold nanostars containing curcumin (R/P-cGNS) and evaluated whether R/P-cGNS had improved anticancer efficacy. We also validated a controlled release profile under near-infrared irradiation for the ability to target melanoma cells and to have an immunomodulatory effect on macrophages. RBC membrane coating provided self-antigens; therefore, it could evade clearance by macrophages, while platelet membrane coating provided targetability to cancer cells. Additionally, the nutraceutical curcumin provided anticancer and anti-inflammatory effects. In conclusion, the results presented in this study demonstrated that R/P-cGNS can deliver drugs to the target region and enhance anticancer effects while avoiding macrophage phagocytosis. We believe that R/P-cGNS can be a new design of the cell-based hybrid system for effective cancer therapy.

Development of ErbB2-Targeting Liposomes for Enhancing Drug Delivery to ErbB2-Positive Breast Cancer.

ErbB2 is a type of receptor tyrosine kinase, which is known to be involved in tumorigenesis, tumor aggressiveness, and clinical outcome. ErbB2-targeting therapy using therapeutic antibodies has been successful in breast cancer treatment. However, the need for repeated treatments and the high cost are major disadvantages with monoclonal antibody therapies. Compared with antibodies, peptides are cheap, relatively stable, and have low immunogenicity. We have developed a highly specific cancer-targeting drug delivery system using a targeting peptide to maximize the therapeutic efficiency of rapamycin and to help prevent drug resistance in ErbB2-positive breast cancer. Physicochemical characterization confirmed the successful construction of ErbB2-targeting liposomes (ErbB2Lipo). A comparison of a scrambled peptide (ScrErbB2) with the ErbB2-targeting peptide confirmed that these peptides had similar properties except for the targeting ability. The ErbB2Lipo exhibited higher delivery efficiency in ErbB2 positive BT-474 cells than non-targeting liposomes conjugated with ScrErbB2 (ScrErbB2Lipo). This peptide-targeting strategy has the potential to improve the efficacy of chemotherapy in ErbB2-positive cancers.

Anomalous Dynamics of in Vivo Cargo Delivery by Motor Protein Multiplexes.

Vesicle transport conducted by motor protein multiplexes (MPMs), which is ubiquitous among eukaryotes, shows anomalous and stochastic dynamics qualitatively different from the dynamics of thermal motion and artificial active matter; the relationship between in vivo vesicle-delivery dynamics and the underlying physicochemical processes is not yet quantitatively understood. Addressing this issue, we perform accurate tracking of individual vesicles, containing upconverting nanoparticles, transported by kinesin-dynein-multiplexes along axonal microtubules. The mean-square-displacement of vesicles along the microtubule exhibits unusual dynamic phase transitions that are seemingly inconsistent with the scaling behavior of the mean-first-passage time over the travel length. These paradoxical results and the vesicle displacement distribution are quantitatively explained and predicted by a multimode MPM model, developed in the current work, where ATP-hydrolysis-coupled motion of MPM has both unidirectional and bidirectional modes.

Lanthanide-Doped Upconversion Nanocarriers for Drug and Gene Delivery.

Compared to traditional cancer treatments, drug/gene delivery is an advanced, safe, and efficient method. Nanoparticles are widely used as nanocarriers in a drug/gene delivery system due to their long circulation time and low multi-drug resistance. In particular, lanthanide-doped upconversion nanoparticles (UCNPs) that can emit UV and visible light by near-infrared (NIR) upconversion demonstrated more efficient and safer drug/gene delivery. Because of the low penetration depth of UV and visible light, a photoinduced reaction such as photocleavage or photoisomerization has proven restrictive. However, NIR light has high tissue penetration depth and stimulates the photoinduced reaction through UV and visible emissions from lanthanide-doped UCNPs. This review discusses the optical properties of UCNPs that are useful in bioapplications and drug/gene delivery systems using the UCNPs as a photoreaction inducer.

Inhibition of glutamine utilization sensitizes lung cancer cells to apigenin-induced apoptosis resulting from metabolic and oxidative stress.

Recent studies have shown anticancer activity of apigenin by suppressing glucose transporter 1 (GLUT1) expression in cultured cancer cells; however, it is not clear whether apigenin can suppress glucose metabolism in lung cancer cells or sensitize them to inhibition of glutamine utilization-mediated apoptosis through metabolic and oxidative stress. We show that apigenin significantly decreases GLUT1 expression in mice. Furthermore, we demonstrate that apigenin induces growth retardation and apoptosis through metabolic and oxidative stress caused by suppression of glucose utilization in lung cancer cells. The underlying mechanisms were defined that the anticancer effects of apigenin were reversed by ectopic GLUT1 overexpression and galactose supplementation, through activation of pentose phosphate pathway-mediated NADPH generation. Importantly, we showed that severe metabolic stress using a glutaminase inhibitor, compound 968, was involved in the mechanism of sensitization by apigenin. Taken together, the combination of apigenin with inhibitors of glutamine metabolism may provide a promising therapeutic strategy for cancer treatment.

Oxidative Dimerization of PHD2 is Responsible for its Inactivation and Contributes to Metabolic Reprogramming via HIF-1α Activation.

Prolyl hydroxylase domain protein 2 (PHD2) belongs to an evolutionarily conserved superfamily of 2-oxoglutarate and Fe(II)-dependent dioxygenases that mediates homeostatic responses to oxygen deprivation by mediating hypoxia-inducible factor-1α (HIF-1α) hydroxylation and degradation. Although oxidative stress contributes to the inactivation of PHD2, the precise molecular mechanism of PHD2 inactivation independent of the levels of co-factors is not understood. Here, we identified disulfide bond-mediated PHD2 homo-dimer formation in response to oxidative stress caused by oxidizing agents and oncogenic H-ras(V12) signalling. Cysteine residues in the double-stranded ß-helix fold that constitutes the catalytic site of PHD isoforms appeared responsible for the oxidative dimerization. Furthermore, we demonstrated that disulfide bond-mediated PHD2 dimerization is associated with the stabilization and activation of HIF-1α under oxidative stress. Oncogenic H-ras(V12) signalling facilitates the accumulation of HIF-1α in the nucleus and promotes aerobic glycolysis and lactate production. Moreover, oncogenic H-ras(V12) does not trigger aerobic glycolysis in antioxidant-treated or PHD2 knocked-down cells, suggesting the participation of the ROS-mediated PHD2 inactivation in the oncogenic H-ras(V12)-mediated metabolic reprogramming. We provide here a better understanding of the mechanism by which disulfide bond-mediated PHD2 dimerization and inactivation result in the activation of HIF-1α and aerobic glycolysis in response to oxidative stress.