Cutting Off H+ Leaks on the Inner Mitochondrial Membrane: A Proton Modulation Approach to Selectively Eradicate Cancer Stem Cells.

Cancer stem cells (CSCs) are associated with the invasion and metastatic relapse of various cancers. However, current cancer therapies are limited to targeting the bulk of primary tumor cells while remaining the CSCs untouched. Here, we report a new proton (H+) modulation approach to selectively eradicate CSCs via cutting off the H+ leaks on the inner mitochondrial membrane (IMM). Based on the fruit extract of Gardenia jasminoides, a multimodal molecule channel blocker with high biosafety, namely, Bo-Mt-Ge, is developed. Importantly, in this study, we successfully identify that mitochondrial uncoupling protein UCP2 is closely correlated with the stemness of CSCs, which may offer a new perspective for selective CSC drug discovery. Mechanistic studies show that Bo-Mt-Ge can specifically inhibit the UCP2 activities, decrease the H+ influx in the matrix, regulate the electrochemical gradient, and deplete the endogenous GSH, which synergistically constitute a unique MoA to active apoptotic CSC death. Intriguingly, Bo-Mt-Ge also counteracts the therapeutic resistance via a two-pronged tactic: drug efflux pump P-glycoprotein downregulation and antiapoptotic factor (e.g., Bcl-2) inhibition. With these merits, Bo-Mt-Ge proved to be one of the safest and most efficacious anti-CSC agents, with ca. 100-fold more potent than genipin alone in vitro and in vivo. This study offers new insights and promising solutions for future CSC therapies in the clinic.

Membrane-Targeting Triphenylphosphonium Functionalized Ciprofloxacin for Methicillin-Resistant Staphylococcus aureus (MRSA).

Multidrug-resistant (MDR) bacteria have become a severe problem for public health. Developing new antibiotics for MDR bacteria is difficult, from inception to the clinically approved stage. Here, we have used a new approach, modification of an antibiotic, ciprofloxacin (CFX), with triphenylphosphonium (TPP, PPh3) moiety via ester- (CFX-ester-PPh3) and amide-coupling (CFX-amide-PPh3) to target bacterial membranes. In this study, we have evaluated the antibacterial activities of CFX and its derivatives against 16 species of bacteria, including MDR bacteria, using minimum inhibitory concentration (MIC) assay, morphological monitoring, and expression of resistance-related genes. TPP-conjugated CFX, CFX-ester-PPh3, and CFX-amide-PPh3 showed significantly improved antibacterial activity against Gram-positive bacteria, Staphylococcus aureus, including MDR S. aureus (methicillin-resistant S. aureus (MRSA)) strains. The MRSA ST5 5016 strain showed high antibacterial activity, with MIC values of 11.12 µg/mL for CFX-ester-PPh3 and 2.78 µg/mL for CFX-amide-PPh3. The CFX derivatives inhibited biofilm formation in MRSA by more than 74.9% of CFX-amide-PPh3. In the sub-MIC, CFX derivatives induced significant morphological changes in MRSA, including irregular deformation and membrane disruption, accompanied by a decrease in the level of resistance-related gene expression. With these promising results, this method is very likely to combat MDR bacteria through a simple TPP moiety modification of known antibiotics, which can be readily prepared at clinical sites.

Mitochondrial relocation of a common synthetic antibiotic: A non-genotoxic approach to cancer therapy.

Tumor recurrence as a result of therapy-induced nuclear DNA lesions is a major issue in cancer treatment. Currently, only a few examples of potentially non-genotoxic drugs have been reported. Mitochondrial re-localization of ciprofloxacin, one of the most commonly prescribed synthetic antibiotics, is reported here as a new approach. Conjugating ciprofloxacin to a triphenyl phosphonium group (giving lead Mt-CFX), is used to enhance the concentration of ciprofloxacin in the mitochondria of cancer cells. The localization of Mt-CFX to the mitochondria induces oxidative damage to proteins, mtDNA, and lipids. A large bias in favor of mtDNA damage over nDNA was seen with Mt-CFX, contrary to classic cancer chemotherapeutics. Mt-CFX was found to reduce cancer growth in a xenograft mouse model and proved to be well tolerated. Mitochondrial relocalization of antibiotics could emerge as a useful approach to generating anticancer leads that promote cell death via the selective induction of mitochondrially-mediated oxidative damage.

Revealing Protein Aggregates under Thapsigargin-Induced ER Stress Using an ER-Targeted Thioflavin.

Endoplasmic reticulum-thioflavin T (ER-ThT), a thioflavin T-based fluorescent chemosensor, was developed to detect protein aggregates in the endoplasmic reticulum (ER) and was applied to live cells under various forms of ER stress. Upon dithiothreitol (DTT)-induced reductive denaturation of lysozyme and albumin, the intensity was increased in a protein concentration-dependent way, following a nonfluorescent lag phase. ER-ThT detects protein aggregates rather than unfolded proteins in solution, and the protein aggregation can be visualized in the presence of lipid membranes or native proteins. Within live HeLa cells, ER-ThT is localized in the ER and its fluorescence was dramatically increased upon ER stress induction by DTT, Thapsigargin, or Brefeldin A. Moreover, in the presence of ER stress modulators (tauroursodeoxycholic acid, trimethylamine N-oxide, or 4-phenylbutyric acid), also known as chemical chaperones, the fluorescence under Thapsigargin treatment was suppressed to the level of the control group. Thus, ER-ThT is capable of detecting the accumulation of protein aggregates under ER stress in living cells and acts as an in vitro screening tool for ER stress modulators, putative prodrugs against ER-related proteopathy. Overall, the results strongly suggest that protein aggregation is intricately involved in the activation of the unfolded protein response following ER stress.

Revisiting Fluorescent Calixarenes: From Molecular Sensors to Smart Materials.

Calix[n]arenes (n = 4, 5, 6, 8) are "chalicelike" phenol-based macrocycles that are among the most fascinating and highly studied scaffolds in supramolecular chemistry. This stems from the functional and tunable diversity at both their upper and lower rims, their preorganized nonpolar cavities and preorganized ion-binding sites, and their well-defined conformations. Conjugation of calixarene scaffolds with various fluorogenic groups has led to the development of smart fluorescent probes that have been utilized as molecular sensors, in bioimaging, for drug and gene delivery, in self-assembly/aggregation, and as smart materials. The fine-tuning and incorporation of different ligating sites in the calix[4]arene scaffold have produced numerous molecular sensors for cations, anions, and biomolecules. Moreover, the aqueous solubility of p-sulfonatocalix[4]arenes has engendered their potential use in drug/gene delivery and enzymatic assays. In addition, because of their strong optical properties, fluorescent calix[4]arenes have been used to develop smart materials, including gels as well as nonlinear optical, organic light-emitting diode, and multiphoton materials. Finally, significant developments in the utility of fluorescent higher calixarenes have been made for bioapplications. This review critically summarizes the recent advances made in all of these different areas.

Nanomaterial designing strategies related to cell lysosome and their biomedical applications: A review.

Lysosomes, an important organelle of eukaryotic cells, are covered with the cell membrane and contain an array of degradative enzymes. The disrupt in lysosomal functions may lead to the development of severe diseases. In nanotechnology, nanomaterials working mechanism and its biomedical output are highly dependent on the lysosomes as it plays a crucial role in intracellular transport. Several nanomaterials specifically designed for lysosome-related actions are highly advantageous in trafficking and delivering the loaded imaging/therapeutic agents. But for other applications, especially gene-based therapeutic delivery into the sub-organelles such as mitochondria and nucleus, lysosomal related degradation could be an obstacle to achieve a maximal therapeutic index. In order to understand the relationship between lysosomes and designed nanomaterials for kind of desired application in biomedical research, complete knowledge of their various designing strategies, size dependent or ligand supportive cellular uptake mechanisms, trafficking, and localizations in eukaryotic cells is highly desired. In this review, we intended to discuss various nanomaterial types that have been applied in biomedical applications based on lysosomal internalization and escape from endo/lysosomes and explored their related advantages/disadvantages. Additionally, we also deliberated nanomaterials direct translocation mechanism, their autophagic accumulation and the underlying mechanism to induced autophagy. Finally, some challenges and critical issues in current research from clinical application perspective has also been addressed. Great understanding of these factors will help in understanding and facilitating the development of safe and effective lysosomal related nanomaterial-based imaging/therapeutic systems for future applications.

Modulating the GSH/Trx selectivity of a fluorogenic disulfide-based thiol sensor to reveal diminished GSH levels under ER stress.

We synthesized a fluorogenic disulfide-based naphthalimide thiol probe (ER-Naph) with a hydrophilic endoplasmic reticulum (ER)-guiding glibenclamide unit. Its ER targeting ability and high selectivity to GSH over thioredoxin, a potent competitor, were clearly demonstrated, both in solution and in vitro. Finally, a confocal microscopic investigation revealed that GSH levels in the ER were dramatically decreased under thapsigargin, brefeldin A, and tunicamycin-induced ER stress models.

A bioorthogonal 'turn-on' fluorescent probe for tracking mitochondrial nitroxyl formation.

A bioreductant-resistant 'turn-on' chemodosimetric fluorescent probe Mito-1 has been developed for the detection of mitochondrial HNO in live cells. Mito-1 enables the detection of HNO as low as ∼18 nM. It has the capability to detect both exogenous and endogenous mitochondrial HNO formations in cellular milieus by providing fluorescence images. Its two-photon imaging ability fosters its use as a noninvasive imaging tool for the detection of mitochondrial nitroxyl.

Highly selective two-photon imaging of cysteine in cancerous cells and tissues.

Abnormal concentrations of Cys have been reported to be implicated in various health problems, including cancer, neuropathy, and cardiomyopathy. We present a novel two-photon fluorescent probe for the specific recognition of cysteine over homocysteine and glutathione, and the bioapplication of this probe for the imaging of live cancerous cells and thick tissues.

Small conjugate-based theranostic agents: an encouraging approach for cancer therapy.

The advances in genomics, proteomics, and bioinformatics have directed the development of new anticancer agents to reduce drug abuse and increase safe and specific drug treatment. Theranostics, combining therapy and diagnosis, is an appealing approach for chemotherapy in medicine which exhibits improved biodistribution, selective cancer targeting ability, reduced toxicity, masked drug efficacy, and minimum side effects. The role of diagnosis tools in theranostics is to collect the information of the diseased state before and after specific treatment. Magnetic particle-, mesoporous silica-, various carbon allotrope-, and polymer nanoparticle-based theranostic systems are well accepted and clinically significant. Currently, small conjugate-based systems have received much attention for cancer treatment and diagnosis. The structural architecture of these systems is relatively simple, compact, biocompatible, and unidirectional. In this tutorial review, we summarize the latest developments on small conjugate based theranostic agents for tumor treatment and diagnosis using fluorescence and magnetic resonance imaging (MRI).

A fluorescent chemosensor for Hg(2+) and Cd(2+) ions in aqueous medium under physiological pH and its applications in imaging living cells.

A new BODIPY derivative with 2,2'-(ethane-1,2-diylbis(oxy))bis(N,N-bis(pyridine-2-ylmethyl)aniline unit as the metal receptor has been designed and synthesized. The dye selectively detects either Cd(2+) or Hg(2+) ions in the presence of hosts of other biologically important and environmentally relevant metal ions in aqueous medium at physiological pH. Binding of metal ions causes a change in the emission behavior of the dye from weakly fluorescent to highly fluorescent. Confocal microscopic experiments validate that the dye can be used to identify changes in either Hg(2+) or Cd(2+) levels in living cells.

The role of copper ions in pathophysiology and fluorescent sensors for the detection thereof.

Copper ions are indispensible to life and maintaining tight control over the homeostasis of copper ions in the body is a prerequisite to sustaining health. Aberrations in normal copper levels, both systemic as well as on a tissue or cellular scale, are implicated in a wide range of diseases, such as Menkes disease, Wilson's disease, Alzheimer's disease, Parkinson's disease and transmissible spongiform encephalopathy (prion diseases). The current understanding of how copper influences these diseases is described. The field of fluorescent copper sensors both functioning via a reaction based mechanism as well as by directly binding copper ions has known an inflation in recent years, and the importance of this field to elucidating the role of copper in cell biology is pointed out. Progress in these tightly interwoven fields has resulted in a better understanding of a number of diseases related to copper imbalances and current developments might open the path for novel and innovating therapies to address these diseases.