Enzyme-Instructed Assemblies Enable Mitochondria Localization of Histone H2B in Cancer Cells

Presently, little is known of how the inter-organelle crosstalk impacts cancer cells owing to the lack of approaches that can manipulate inter-organelle communication in cancer cells. We found that a negatively charged, enzyme cleavable peptide (MitoFlag) enables the trafficking of histone protein H2B, a nuclear protein, to the mitochondria in cancer cells. MitoFlag interacts with the nuclear location sequence of H2B to block it from entering the nucleus. A protease on the mitochondria cleaves the Flag from the MitoFlag/H2B complex to form assemblies that retain H2B on the mitochondria and facilitate H2B entering the mitochondria. Adding NLS, replacing aspartic acid by glutamic acid residues, or changing the l - to d -aspartic acid residue on MitoFlag abolishes the trafficking of H2B into mitochondria of HeLa cells. As the first example of the enzyme-instructed self-assembly of a synthetic peptide for trafficking endogenous proteins, this work provides insights for understanding and manipulating inter-organelle communication in cells.     

Mitochondria‐Targeted Cancer Therapy Using a Light‐Up Probe with Aggregation‐Induced‐Emission Characteristics

Subcellular organelle‐specific reagents for simultaneous tumor targeting, imaging, and treatment are of enormous interest in cancer therapy. Herein, we present a mitochondria‐targeting probe (AIE‐mito‐TPP) by conjugating a triphenylphosphine (TPP) with a fluorogen which can undergo aggregation‐induced emission (AIE). Owing to the more negative mitochondrial membrane potential of cancer cells than normal cells, the AIE‐mito‐TPP probe can selectively accumulate in cancer‐cell mitochondria and light up its fluorescence. More importantly, the probe exhibits selective cytotoxicity for studied cancer cells over normal cells. The high potency of AIE‐mito‐TPP correlates with its strong ability to aggregate in mitochondria, which can efficiently decrease the mitochondria membrane potential and increase the level of intracellular reactive oxygen species (ROS) in cancer cells. The mitochondrial light‐up probe provides a unique strategy for potential image‐guided therapy of cancer cells.     

Tetraphenylethylene Conjugated with a Specific Peptide as a Fluorescence Turn‐On Bioprobe for the Highly Specific Detection and Tracing of Tumor Markers in Live Cancer Cells

Smart molecular probes and flexible methods are attracting remarkable interest for the visualization of cancer‐related biological and chemical events. In this work, a new fluorescence turn‐on probe with dual‐recognition characteristics for the specific imaging of cancer cells is reported. This new bioprobe is rationally designed by linking tetraphenylethylene (TPE), an aggregation‐induced emission (AIE) fluorophore, with the small peptide IHGHHIISVG (referred to as AP2H), a targeting ligand to the broad‐spectrum cancer‐related protein LAPTM4B. The binding of the probe TPE‐AP2H with the target, both in solution and at the cellular level, switches on the fluorescence of TPE because of the inhibition of internal rotations within the TPE framework. Accordingly, this bioprobe allows the real‐time monitoring and subcellular localization of LAPTM4B in cancer cells, with a very high target‐to‐background ratio for the imaging. Furthermore, brighter fluorescence images are detected after incubation of TPE‐AP2H with tumor cells at lower pH values. Thus, this new bioprobe is more advantageous because it can simultaneously target the LAPTM4B protein and sense the characteristic low‐pH environment of tumor cells. In addition, TPE‐AP2H displays high photostability and low cytotoxicity. Therefore, this new bioprobe is promising for the more accurate and reliable imaging of tumor markers in live cancer cells.     

Cisplatin‐Rich Polyoxazoline–Poly(aspartic acid) Supramolecular Nanoparticles

Cisplatin‐rich supramolecular nanoparticles are constructed through the supramolecular inclusion interaction between the admantyl (Ad)‐terminated poly(aspartic acid) (Ad‐P(Asp)) and the β‐cyclodextrin (β‐CD)‐terminated poly(2‐methyl‐2‐oxazoline). In the formation of the nanoparticles, the β‐CD/admantane inclusion complex integrates poly(2‐methyl‐2‐oxazoline) and poly(aspartic acid) chains to form pseudoblock copolymers, followed by the coordination between carboxyl groups in P(Asp) block and cisplatin. This coordination interaction drives the formation of nanoparticle and enables cisplatin incorporated into the nanoparticles. The spherical cisplatin‐rich supramolecular nanoparticles have 53% cisplatin‐loading content, good stability, and effective inhibition of the cell proliferation when it is tested in H22 cancer cells. Near‐infrared fluorescence imaging of tumor bearing mice reveals that the cisplatin‐rich nanoparticles can target the tumor in vivo effectively.     

Oligonucleotide loaded polypeptide‐peptide nanoparticles towards mitochondrial‐targeted delivery

Nanoparticles (NPs) consisting of biodegradable and biocompatible polymers may have the ability to deliver a cargo to specific tissue, cell type, and organelle. Various diseases, which are linked to mitochondrial genome (mtDNA) mutations and have no effective treatments, may be approached by gene therapy strategies. In this study, we adapted the recently developed mitochondria delivering polypeptide‐peptide nanoparticles (PoP‐NPs) system to carry an oligonucleotide cargo to the proximity of the mitochondria. PoP‐NPs are formulated by self‐assembly of the negatively charged polypeptide, poly gamma glutamic acid (γ‐PGA), with an amphiphilic and cationic β‐sheet peptide (PFK). Here, we show that PFK interacts favorably with oligonucleotides and thereby enables the formation of DNA‐loaded PoP‐NPs (DNA‐PoP‐NPs). DNA‐PoP‐NPs could be assembled with different peptide to oligonucleotide (N/P) ratios, and their targeting to the proximity of mitochondria in cell culture could be facilitated through NPs coating with PFK peptide.     

Cathepsin‐B Induced Controlled Release from Peptide‐Capped Mesoporous Silica Nanoparticles

New capped silica mesoporous nanoparticles for intracellular controlled cargo release within cathepsin B expressing cells are described. Nanometric mesoporous MCM‐41 supports loaded with safranin O ( S1‐P ) or doxorubicin ( S2‐P ) containing a molecular gate based on a cathepsin B target peptidic sequence were synthesized. Solids were designed to show “zero delivery” and to display cargo release in the presence of cathepsin B enzyme, which selectively hydrolyzed in vitro the capping peptide sequence. Controlled delivery in HeLa, MEFs WT, and MEFs lacking cathepsin B cell lines were also tested. Release of safranin O and doxorubicin in these cells took place when cathepsin B was active or present. Cells treated with S2‐P showed a fall in cell viability due to nanoparticles internalization, cathepsin B hydrolysis of the capping peptide, and cytotoxic agent delivery, proving the possible use of these nanodevices as new therapeutic tools for cancer treatment.     

Bio‐Orthogonal Polymer Coatings for Co‐Presentation of Biomolecules

Controlled presentation of biomolecules on synthetic substrates is an important aspect for biomaterials development. If the immobilization of multiple biomolecules is required, highly efficient orthogonal surface chemistries are needed to ensure the precision of the immobilization. In this communication, chemical vapor deposition (CVD) copolymerization is used to fabricate polymer coatings with controlled ratio of alkyne and pentafluorophenyl ester (Pfp‐ester) groups. Cyclic argine‐glycine‐aspartic acid (cRGD) adhesion peptide and epidermal growth factor (EGF) are immobilized through alkyne–azide cycloaddtion (“click” chemistry) and active ester–amine reaction, respectively. Cell studies with human umbilical vein endothelial cells (HUVEC) and A431 cell lines demonstrate the biological activity of the coimmobilized biomolecules.     

Independent highly sensitive characterization of asparagine deamidation and aspartic acid isomerization by sheathless CZE‐ESI‐MS/MS

Amino acids residues are commonly submitted to various physicochemical modifications occurring at physiological pH and temperature. Post‐translational modifications (PTMs) require comprehensive characterization because of their major influence on protein structure and involvement in numerous in vivo process or signaling. Mass spectrometry (MS) has gradually become an analytical tool of choice to characterize PTMs; however, some modifications are still challenging because of sample faint modification levels or difficulty to separate an intact peptide from modified counterparts before their transfer to the ionization source. Here, we report the implementation of capillary zone electrophoresis coupled to electrospray ionization tandem mass spectrometry (CZE‐ESI‐MS/MS) by the intermediate of a sheathless interfacing for independent and highly sensitive characterization of asparagine deamidation (deaN) and aspartic acid isomerization (isoD). CZE selectivity regarding deaN and isoD was studied extensively using different sets of synthetic peptides based on actual tryptic peptides. Results demonstrated CZE ability to separate the unmodified peptide from modified homologous exhibiting deaN, isoD or both independently with a resolution systematically superior to 1.29. Developed CZE‐ESI‐MS/MS method was applied for the characterization of monoclonal antibodies and complex protein mixture. Conserved CZE selectivity could be demonstrated even for complex samples, and foremost results obtained showed that CZE selectivity is similar regardless of the composition of the peptide. Separation of modified peptides prior to the MS analysis allowed to characterize and estimate modification levels of the sample independently for deaN and isoD even for peptides affected by both modifications and, as a consequence, enables to distinguish the formation of l ‐aspartic acid or d ‐aspartic acid generated from deaN. Separation based on peptide modification allowed, as supported by the ESI efficiency provided by CZE‐ESI‐MS/MS properties, and enabled to characterize and estimate studied PTMs with an unprecedented sensitivity and proved the relevance of implementing an electrophoretic driven separation for MS‐based peptide analysis. Copyright © 2016 John Wiley & Sons, Ltd.     

Poly(ethyleneimine)/arginine–glycine–aspartic acid conjugates prepared with N‐succinimidyl 3‐(2‐pyridyldithio)propionate: An investigation of peptide coupling and conjugate stability

Poly(ethylene imine) (PEI), a highly cationic polymer, is being used for deoxyribonucleic acid (DNA) complexation and delivery into cells. To enhance the cellular uptake of polymer/DNA complexes, arginine–glycine–aspartic acid (RGD) peptides have been conjugated to PEI with N‐succinimidyl 3‐(2‐pyridyldithio)propionate (SPDP). This coupling scheme creates a disulfide‐linked conjugate, the stability of which in the presence of thiols is uncertain. We have investigated the conjugation of an RGD peptide, glycine–arginine–glycine–aspartic acid–serine–proline–cysteine (GRGDSPC), to PEI with SPDP and subsequently assessed the stability of the conjugates in the presence of two thiol compounds, mercaptoethanol and cysteine. SPDP effectively controls the extent of GRGDSPC substitution on PEI. The conjugates, however, are readily cleaved in the presence of the thiols; the cleavage is rapid (～50% cleavage in 2–4 h) and inversely related to the degree of peptide substitution on the polymers. The peptide coupling is stable in the absence of thiols, and its cleavage is strongly dependent on the pH of the medium but not on the ionic strength of the medium. We conclude that RGD peptides coupled to PEI are labile in the presence of physiological concentrations of thiols, and this should be taken into account when such polymer–peptide conjugates are used for DNA delivery. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 6143–6156, 2004     

Enzymatically Formed Peptide Assemblies Sequestrate Proteins and Relocate Inhibitors to Selectively Kill Cancer Cells

Herein, we show that an enzymatic reaction can generate peptide assemblies that sequestrate proteins to selectively kill cancer cells. A phosphopeptide bearing the antagonistic motif (AVPI) to the inhibitors of apoptotic proteins (IAPs) enters cancer cells and normal cells by caveolin‐dependent endocytosis and macropinocytosis, respectively. The AVPI‐bearing peptide assemblies sequestrates IAPs and releases bortezomib (BTZ), a proteasome inhibitor, in the cytosol of cancer cells, but rescues the normal cells (namely, HS‐5 cells) by trafficking the BTZ into lysosomes. Alkaline phosphatase (ALP) acts as a context‐dependent signal for trafficking the peptide/BTZ assemblies and selectively induces the death of the cancer cells. The assemblies of AVPI exhibit enhanced proteolytic resistance. This work, which utilizes the difference in endocytic uptake of enzymatically formed peptide assemblies to selectively kill cancer cells, promises a new way to develop selective cancer therapeutics.     

Peptide‐Coated Platinum Nanoparticles with Selective Toxicity against Liver Cancer Cells

Peptide‐stabilized platinum nanoparticles (PtNPs) were developed that have significantly greater toxicity against hepatic cancer cells (HepG2) than against other cancer cells and non‐cancerous liver cells. The peptide H‐Lys‐Pro‐Gly‐d Lys‐NH₂ was identified by a combinatorial screening and further optimized to enable the formation of water‐soluble, monodisperse PtNPs with average diameters of 2.5 nm that are stable for years. In comparison to cisplatin, the peptide‐coated PtNPs are not only more toxic against hepatic cancer cells but have a significantly higher tumor cell selectivity. Cell viability and uptake studies revealed that high cellular uptake and an oxidative environment are key for the selective cytotoxicity of the peptide‐coated PtNPs.     

Conformation and structure of bis­(glycyl‐l‐aspartic acid) oxalate 0.4‐hydrate

The title bis­(glycyl‐l ‐aspartic acid) oxalate complex {systematic name: bis­[2‐(2‐ammonio­acetamido)butane­dioic acid] oxalate 0.4‐hydrate}, 2C₆H₁₁N₂O₅⁺·C₂O₄²⁻·4H₂O, crystallizes in a triclinic space group with the planar peptide unit in a trans conformation. The asymmetric unit consists of two glycyl‐l ‐aspartic acid mol­ecules with positively charged amino groups and neutral carboxyl groups, and an oxalate dianion. The twist around the C—Cα bond indicates that both the peptide mol­ecules adopt extended conformations, while the twist around the N—Cα bond shows that one has a folded and the other a semi‐extended state. The present complex can be described as an inclusion compound with the dipeptide mol­ecule as the host and the oxalate anion as the guest. The usual head‐to‐tail sequence of aggregation is not observed in this complex, as is also the case with the glycyl‐l ‐aspartic acid dihydrate mol­ecule. The study of aggregation and inter­action patterns in binary systems is the first step towards understanding more complex phenomena. This further leads to results that are of general interest in bimolecular aggregation.     

Organelle‐Targeted BODIPY Photocages: Visible‐Light‐Mediated Subcellular Photorelease

Photocaging facilitates non‐invasive and precise spatio‐temporal control over the release of biologically relevant small‐ and macro‐molecules using light. However, sub‐cellular organelles are dispersed in cells in a manner that renders selective light‐irradiation of a complete organelle impractical. Organelle‐specific photocages could provide a powerful method for releasing bioactive molecules in sub‐cellular locations. Herein, we report a general post‐synthetic method for the chemical functionalization and further conjugation of meso‐methyl BODIPY photocages and the synthesis of endoplasmic reticulum (ER)‐, lysosome‐, and mitochondria‐targeted derivatives. We also demonstrate that 2,4‐dinitrophenol, a mitochondrial uncoupler, and puromycin, a protein biosynthesis inhibitor, can be selectively photoreleased in mitochondria and ER, respectively, in live cells by using visible light. Additionally, photocaging is shown to lead to higher efficacy of the released molecules, probably owing to a localized and abrupt release.     

Carboplatin‐Complexed and cRGD‐Conjugated Unimolecular Nanoparticles for Targeted Ovarian Cancer Therapy

Platinum‐based chemotherapy has been widely used to treat cancers including ovarian cancer; however, it suffers from dose‐limiting toxicity. Judiciously designed drug nanocarriers can enhance the anticancer efficacy of platinum‐based chemotherapy while reducing its systemic toxicity. Herein the authors report a stable and water‐soluble unimolecular nanoparticle constructed from a hydrophilic multi‐arm star block copolymer poly(amidoamine)‐b‐poly(aspartic acid)‐b‐poly(ethylene glycol) (PAMAM‐PAsp‐PEG) conjugated with both cRGD (cyclo(Arg‐Gly‐Asp‐D‐Phe‐Cys) peptide and cyanine5 (Cy5) fluorescent dye as a platinum‐based drug nanocarrier for targeted ovarian cancer therapy. Carboplatin is complexed to the poly(aspartic acid) inner shell via pH‐responsive ion–dipole interactions between carboplatin and the carboxylate groups of poly(aspartic acid). Based on flow cytometry and confocal laser scanning microscopy analyses, cRGD‐conjugated unimolecular nanoparticles exhibit much higher cellular uptake by ovarian cancer cells overexpressing α_vβ₃ integrin than nontargeted (i.e., cRGD‐lacking) ones. Carboplatin‐complexed cRGD‐conjugated nanoparticles also exhibit higher cytotoxicity than nontargeted nanoparticles as well as free carboplatin, while empty unimolecular nanoparticles show no cytotoxicity. These results indicate that stable unimolecular nanoparticles made of individual hydrophilic multi‐arm star block copolymer molecules conjugate with tumor‐targeting ligands and dyes (i.e., PAMAM‐PAsp‐PEG‐cRGD/Cy5) are promising nanocarriers for platinum‐based anticancer drugs for targeted cancer therapy.

     

A Multi‐responsive Fluorescent Probe Reveals Mitochondrial Nucleoprotein Dynamics with Reactive Oxygen Species Regulation through Super‐resolution Imaging

Understanding the biomolecular interactions in a specific organelle has been a long‐standing challenge because it requires super‐resolution imaging to resolve the spatial locations and dynamic interactions of multiple biomacromolecules. Two key difficulties are the scarcity of suitable probes for super‐resolution nanoscopy and the complications that arise from the use of multiple probes. Herein, we report a quinolinium derivative probe that is selectively enriched in mitochondria and switches on in three different fluorescence modes in response to hydrogen peroxide (H₂O₂), proteins, and nucleic acids, enabling the visualization of mitochondrial nucleoprotein dynamics. STED nanoscopy reveals that the proteins localize at mitochondrial cristae and largely fuse with nucleic acids to form nucleoproteins, whereas increasing H₂O₂ level leads to disassociation of nucleic acid–protein complexes.     

A Combined Effect of Thermal and Enzymatic Racemization for d‐Aspartic Acid to l‐Aspartic Acid by High‐Performance Liquid Chromatography Assay

The racemization of d ‐aspartic acid to l ‐aspartic acid has been successfully performed with a coupled enzyme system at 90 °C and a pH of about 4.0 by the assay of high‐performance liquid chromatography. This coupled enzymatic racemization is a successive two‐step reaction first induced by d ‐amino acid oxidase and a subsequent coupled reaction by an aminotransferase clonezyme with the help of coenzyme pyridoxal 5′‐phosphate and cosubstrate l ‐glutamate. Due to the very high temperature, part of the l ‐aspartic acid is produced by the thermal effect. In fact the thermal racemization for aspartic acid can proceed from either d ‐ or l ‐aspartic acid via an intermediate fumaric acid and leads to the formation of d ,l ‐malic acid. The formation of α‐oxalacetic acid formed irreversibly from d ‐aspartic acid with d ‐amino acid oxidase can induce a side reaction to l ‐alanine. The thermal effect may also be responsible for the production of d ‐, and l ‐alanine.     

Biological Glucose Metabolism Regulated Peptide Self‐Assembly as a Simple Visual Biosensor for Glucose Detection

A glucose oxidase (GOx)‐mediated glucose metabolism was in vitro mimicked and employed to regulate the self‐assembly of peptide‐based building blocks. In this new stimuli‐responsive self‐assembly system, two peptide‐based building blocks, respectively, having aspartic acid (gelator 1 ) and lysine (gelator 2 ) residues were designed and prepared. When adding glucose and GOx to the aqueous solution of gelator 1 or the self‐assembled fibrillar hydrogel of gelator 2 to construct glucose metabolism system, the metabolic product (gluconic acid) can trigger the protonation of the peptide molecules and induce the phase transitions of gelators 1 (sol‐gel) and 2 (gel‐sol). Because this glucose metabolism regulated peptide self‐assembly is built on the oxidation of glucose, it can be used as a simple visual biosensor for glucose detection.     

A Click Cage: Organelle‐Specific Uncaging of Lipid Messengers

Lipid messengers exert their function on short time scales at distinct subcellular locations, yet most experimental approaches for perturbing their levels trigger cell‐wide concentration changes. Herein, we report on a coumarin‐based photocaging group that can be modified with organelle‐targeting moieties by click chemistry and thus enables photorelease of lipid messengers in distinct organelles. We show that caged arachidonic acid and sphingosine derivatives can be selectively delivered to mitochondria, the ER, lysosomes, and the plasma membrane. By comparing the cellular calcium transients induced by localized uncaging of arachidonic acid and sphingosine, we show that the precise intracellular localization of the released second messenger is crucial for the signaling outcome. Ultimately, we anticipate that this new class of caged compounds will greatly facilitate the study of cellular processes on the organelle level.     

Synthesis and Conformational Studies of a Hybrid Cyclic Peptide Based on cis‐β‐Furanoid Sugar Amino Acid (FSAA) and Ornithine

The conformational control of a 14‐helix nucleating template, cis‐β‐furanoid sugar amino acid (FSAA), over a flexible δ‐amino acid, ornithine is studied in a FSAA‐ornithine cyclic tetrapeptide. Extensive NMR and MD studies reveal that the cyclic peptide adopts a three‐dimentional bowl‐shape cavity, which promotes six‐ and seven‐membered intra‐ and inter‐residue H‐bonding, in polar and non‐polar solvents, respectively.