Real-Time Multi-Photon Tracking and Bioimaging of Glycosylated Theranostic Prodrugs upon Specific Enzyme Triggered Release.

Real-time tracking of prodrug uptake, delivery and activation in vivo represents a major challenge for prodrug development. Herein, we demonstrate the use of novel glycosylated theranostics of the cancer pharmacophore Amonafide in highly-selective, enzymatic triggered release. We show that the use of endogenous enzymes for activated release of the therapeutic component can be observed, in real time, and monitored using one and two-photon bioimaging, offering unique insight into the prodrug pharmacokinetic profile. Furthermore, we demonstrate that the potent cytotoxicity of Amonafide is preserved using this targeted approach.

Design, Synthesis, and Anticancer Studies of a p-Cymene-Ru(II)-Curcumin Organometallic Conjugate Based on a Fluorescent 4-Amino-1,8-naphthalimide Tröger's Base Scaffold.

A unique V-shaped "chiral" supramolecular scaffold, N-(4-pyridyl)-4-amino-1,8-naphthalimide Tröger's base (TBNap), was synthesized in good yield from a precursor N-(4-pyridyl)-4-amino-1,8-naphthalimide (Nap). TBNap was characterized using different spectroscopic methods and the molecular structure was elucidated by diffraction analysis. A new p-cymene-Ru(II)-curcumin conjugate (TB-Ru-Cur) was designed by reacting TBNap dipyridyl donor and ruthenium-curcuminato acceptor [RuCur = (p-cymene)Ru-(curcuminato)Cl] in the presence of silver triflate. TB-Ru-Cur was isolated in quantitative yield and characterized using Fourier transform infrared (FT-IR), NMR (1H, 13C, and 19F), and electrospray ionization mass spectrometry (ESI-MS), and the molecular structure has been predicted using a computational study. Both TBNap and TB-Ru-Cur exhibited intramolecular charge transfer (ICT)-based fluorescence emission. Furthermore, the anticancer properties of TBNap, Ru-Cur, and TB-Ru-Cur were assessed in different cancer cell lines. Gratifyingly, the conjugate TB-Ru-Cur displayed fast-cellular internalization and good cytotoxicity against HeLa, HCT-116, and HepG2 cancer cells and the estimated IC50 value was much lower than that of the precursors (TBNap and Ru-Cur) and the well-known chemotherapeutic drug cisplatin.

Functionalization of carbon nanoparticles modulates inflammatory cell recruitment and NLRP3 inflammasome activation.

The inflammatory effects of carbon nanoparticles (NPs) are highly disputed. Here it is demonstrated that endotoxin-free preparations of raw carbon nanotubes (CNTs) are very limited in their capacity to promote inflammatory responses in vitro, as well as in vivo. Upon purification and selective oxidation of raw CNTs, a higher dispersibility is achieved in physiological solutions, but this process also enhances their inflammatory activity. In synergy with toll-like receptor (TLR) ligands, CNTs promote NLRP3 inflammasome activation and it is shown for the first time that this property extends to spherical carbon nano-onions (CNOs) of 6 nm in size. In contrast, the benzoic acid functionalization of purified CNTs and CNOs leads to significantly attenuated inflammatory properties. This is evidenced by a reduced secretion of the inflammatory cytokine IL-1ß, and a pronounced decrease in the recruitment of neutrophils and monocytes following injection into mice. Collectively, these results reveal that the inflammatory properties of carbon NPs are highly dependent on their physicochemical characteristics and crucially, that chemical surface functionalization allows significant moderation of these properties.

Effect of Alkyl Chain Length on the Photophysical, Photochemical, and Photobiological Properties of Ruthenium(II) Polypyridyl Complexes for Their Application as DNA-Targeting, Cellular-Imaging, and Light-Activated Therapeutic Agents.

A family of six Ru(II) polypyridyl complexes (1-6) which contain phenanthroline-based ligands functionalized with alkyl chains of different lengths (one methyl group, 10 and 21 carbon alkyl chains) and either 1,10-phenanthroline (phen) or 1,4,5,8-tetraazaphenanthrene (TAP) as ancillary ligands have been synthesized and characterized. The influence of the alkyl chain length on their photophysical and photochemical properties as well as in their photobiological applications has been elucidated by monitoring the changes in their MLCT-centered absorption and emission bands. The presence of one methyl group or 10 carbon alkyl chains does not seem to significantly affect the photophysical and photochemical properties of the resulting Ru(II) complexes when compared to the well-known [Ru(phen)3]2+ and [Ru(TAP)2phen]2+. However, an effect on their emission properties and in their ability to photosensitize singlet oxygen is observed for the Ru(II) complexes containing 21 carbon alkyl chains. The binding of these complexes to salmon testes DNA (stDNA) was investigated by observing the changes in the photophysical properties. Complexes 1, 2, 4, and 5 all showed changes in their MLCT bands that could be analyzed using conventional fitting methods, such as the Bard equation. In contrast, complexes 3 and 6, possessing long aliphatic chains, gave rise to nonclassic behavior. In addition to these analyses, both thermal denaturation and circular dichroism studies of 1-6 were carried out in the presence of stDNA which confirmed that these complexes bind to DNA. Confocal microscopy and viability studies in HeLa cervical cancer cells reveal an alkyl chain-length dependence on the cellular uptake and cytotoxicity of the resulting Ru(II) complexes due to an enhancement of their lipophilicity with increasing alkyl chain length. Thus, complexes containing 10 and 21 carbon alkyl chains are rapidly taken up into HeLa cells and, in particular, those with 21 carbon alkyl chains show a significant phototoxicity against the same cell line. Therefore, this study provides further insight into the possible modulation of the photophysical, photochemical, and photobiological properties of Ru(II) polypyridyl complexes by varying the length of the alkyl chains attached to the polypyridyl ligands coordinated to the Ru(II) center and the nature of the auxiliary groups, which we show has a significant effect on photophysical and biological properties.

Functionalisation of gold nanoparticles with ruthenium(II) polypyridyl complexes for their application in cellular imaging.

Two new dinuclear Ru(ii) polypyridyl complexes containing an alkyl disulphide functionalised bipyridine-based ligand and either 1,10-phenanthroline (phen) or 1,4,5,8-tetraazaphenanthrene (TAP) as ancillary ligands have been synthesised and characterised. Their attachment onto the surface of gold nanoparticles (AuNPs, average diameter of ca. 2.5 nm) resulted in the formation of two new water-soluble Ru(ii)-AuNP conjugates that combine the advantageous properties of both moieties. Both free complexes show the attractive photophysical properties of Ru(ii) polypyridyl complexes and a rapid cellular uptake in HeLa cervical cancer cells. However, their corresponding gold conjugates displayed lower quantum yields than those determined for the free complexes presumed to be due to an energy transfer quenching of the Ru(ii) luminescence by interaction with the gold surface. Despite their diminished luminescence, confocal fluorescence microscopy studies revealed that the Ru(ii)-AuNP conjugates are successfully internalised into HeLa cells and better tolerated than their free complex counterparts after 24 h incubation, which makes them potential luminescent nanomaterials for bioimaging applications.

Water-soluble amphiphilic ruthenium(ii) polypyridyl complexes as potential light-activated therapeutic agents.

Two water-soluble amphiphilic Ru(ii) polypyridyl complexes containing N-1,10-phenanthrolin-5-yldocosanamide and 1,10-phenanthroline (phen) or 1,4,5,8-tetraazaphenanthrene (TAP) as ligands were synthesised and their photophysical and photobiological properties evaluated; both complexes showed a rapid cellular uptake and displayed phototoxicity against HeLa cervical cancer cells.

Genetic Deletion of Zebrafish Rab28 Causes Defective Outer Segment Shedding, but Not Retinal Degeneration.

The photoreceptor outer segment is the canonical example of a modified and highly specialized cilium, with an expanded membrane surface area in the form of disks or lamellae for efficient light detection. Many ciliary proteins are essential for normal photoreceptor function and cilium dysfunction often results in retinal degeneration leading to impaired vision. Herein, we investigate the function and localization of the ciliary G-protein RAB28 in zebrafish cone photoreceptors. CRISPR-Cas9 generated rab28 mutant zebrafish display significantly reduced shed outer segment material/phagosomes in the RPE at 1 month post fertilization (mpf), but otherwise normal visual function up to 21 dpf and retinal structure up to 12 mpf. Cone photoreceptor-specific transgenic reporter lines show Rab28 localizes almost exclusively to outer segments, independently of GTP/GDP nucleotide binding. Co-immunoprecipitation analysis demonstrates tagged Rab28 interacts with components of the phototransduction cascade, including opsins, phosphodiesterase 6C and guanylate cyclase 2D. Our data shed light on RAB28 function in cones and provide a model for RAB28-associated cone-rod dystrophy.

Primary Cilium Formation and Ciliary Protein Trafficking Is Regulated by the Atypical MAP Kinase MAPK15 in Caenorhabditis elegans and Human Cells.

Motile and immotile (or primary) cilia are microtubule-based structures that mediate multiple cellular functions, including the transduction of environmental cues, developmental signaling, cellular motility, and modulation of fluid flow. Although their core architectures are similar, motile and primary cilia exhibit marked structural differences that underlie distinct functional properties. However, the extent to which ciliogenesis mechanisms are shared between these different cilia types is not fully described. Here, we report that the atypical MAP kinase MAPK15 (ERK7/8), implicated in the formation of vertebrate motile cilia, also regulates the formation of primary cilia in Caenorhabditis elegans sensory neurons and human cells. We find that MAPK15 localizes to a basal body subdomain with the ciliopathy protein BBS7 and to cell-cell junctions. MAPK15 also regulates the localization of ciliary proteins involved in cilium structure, transport, and signaling. Our results describe a primary cilia-related role for this poorly studied member of the MAPK family in vivo, and indicate a broad requirement for MAPK15 in the formation of multiple ciliary classes across species.

TMEM107 recruits ciliopathy proteins to subdomains of the ciliary transition zone and causes Joubert syndrome.

The transition zone (TZ) ciliary subcompartment is thought to control cilium composition and signalling by facilitating a protein diffusion barrier at the ciliary base. TZ defects cause ciliopathies such as Meckel-Gruber syndrome (MKS), nephronophthisis (NPHP) and Joubert syndrome (JBTS). However, the molecular composition and mechanisms underpinning TZ organization and barrier regulation are poorly understood. To uncover candidate TZ genes, we employed bioinformatics (coexpression and co-evolution) and identified TMEM107 as a TZ protein mutated in oral-facial-digital syndrome and JBTS patients. Mechanistic studies in Caenorhabditis elegans showed that TMEM-107 controls ciliary composition and functions redundantly with NPHP-4 to regulate cilium integrity, TZ docking and assembly of membrane to microtubule Y-link connectors. Furthermore, nematode TMEM-107 occupies an intermediate layer of the TZ-localized MKS module by organizing recruitment of the ciliopathy proteins MKS-1, TMEM-231 (JBTS20) and JBTS-14 (TMEM237). Finally, MKS module membrane proteins are immobile and super-resolution microscopy in worms and mammalian cells reveals periodic localizations within the TZ. This work expands the MKS module of ciliopathy-causing TZ proteins associated with diffusion barrier formation and provides insight into TZ subdomain architecture.