Reproducible Synthesis of Biocompatible Albumin Nanoparticles Designed for Intra-articular Administration of Celecoxib to Treat Osteoarthritis.

Osteoarthritis (OA) is the most common form of arthritis, with intra-articular (IA) delivery of therapeutics being the current best option to treat pain and inflammation. However, IA delivery is challenging due to the rapid clearance of therapeutics from the joint and the need for repeated injections. Thus, there is a need for long-acting delivery systems that increase the drug retention time in joints with the capacity to penetrate OA cartilage. As pharmaceutical utility also demands that this is achieved using biocompatible materials that provide colloidal stability, our aim was to develop a nanoparticle (NP) delivery system loaded with the COX-2 inhibitor celecoxib that can meet these criteria. We devised a reproducible and economical method to synthesize the colloidally stable albumin NPs loaded with celecoxib without the use of any of the following conditions: high temperatures at which albumin denaturation occurs, polymer coatings, oils, Class 1/2 solvents, and chemical protein cross-linkers. The spherical NP suspensions were biocompatible, monodisperse with average diameters of 72 nm (ideal for OA cartilage penetration), and they were stable over 6 months at 4 °C. Moreover, the NPs loaded celecoxib at higher levels than those required for the therapeutic response in arthritic joints. For these reasons, they are the first of their kind. Labeled NPs were internalized by primary human articular chondrocytes cultured from the knee joints of OA patients. The NPs reduced the concentration of inflammatory mediator prostaglandin E2 released by the primaries, an indication of retained bioactivity following NP synthesis. Similar results were observed in lipopolysaccharide-stimulated human THP-1 monocytes. The IA administration of these NPs is expected to avoid side-effects associated with oral administration of celecoxib and to maintain a high local concentration in the knee joint over a sustained period. They are now ready for evaluation by IA administration in animal models of OA.

Good Vibrations Report on the DNA Quadruplex Binding of an Excited State Amplified Ruthenium Polypyridyl IR Probe.

The nitrile containing Ru(II)polypyridyl complex [Ru(phen)2(11,12-dCN-dppz)]2+ (1) is shown to act as a sensitive infrared probe of G-quadruplex (G4) structures. UV-visible absorption spectroscopy reveals enantiomer sensitive binding for the hybrid htel(K) and antiparallel htel(Na) G4s formed by the human telomer sequence d[AG3(TTAG3)3]. Time-resolved infrared (TRIR) of 1 upon 400 nm excitation indicates dominant interactions with the guanine bases in the case of Λ-1/htel(K), Δ-1/htel(K), and Λ-1/htel(Na) binding, whereas Δ-1/htel(Na) binding is associated with interactions with thymine and adenine bases in the loop. The intense nitrile transient at 2232 cm-1 undergoes a linear shift to lower frequency as the solution hydrogen bonding environment decreases in DMSO/water mixtures. This shift is used as a sensitive reporter of the nitrile environment within the binding pocket. The lifetime of 1 in D2O (ca. 100 ps) is found to increase upon DNA binding, and monitoring of the nitrile and ligand transients as well as the diagnostic DNA bleach bands shows that this increase is related to greater protection from the solvent environment. Molecular dynamics simulations together with binding energy calculations identify the most favorable binding site for each system, which are in excellent agreement with the observed TRIR solution study. This study shows the power of combining the environmental sensitivity of an infrared (IR) probe in its excited state with the TRIR DNA "site effect" to gain important information about the binding site of photoactive agents and points to the potential of such amplified IR probes as sensitive reporters of biological environments.

Correction: Time-resolved infra-red studies of photo-excited porphyrins in the presence of nucleic acids and in HeLa tumour cells: insights into binding site and electron transfer dynamics.

Correction for 'Time-resolved infra-red studies of photo-excited porphyrins in the presence of nucleic acids and in HeLa tumour cells: insights into binding site and electron transfer dynamics' by Páraic M. Keane et al., Phys. Chem. Chem. Phys., 2022, 24, 27524-27531, https://doi.org/10.1039/D2CP04604K.

G-Quadruplex Binding of an NIR Emitting Osmium Polypyridyl Probe Revealed by Solution NMR and Time-Resolved Infrared Studies.

G-quadruplexes are emerging targets in cancer research and understanding how diagnostic probes bind to DNA G-quadruplexes in solution is critical to the development of new molecular tools. In this study the binding of an enantiopure NIR emitting [Os(TAP)2 (dppz)]2+ complex to different G-quadruplex structures formed by human telomer (hTel) and cMYC sequences in solution is reported. The combination of NMR and time-resolved infrared spectroscopic techniques reveals the sensitivity of the emission response to subtle changes in the binding environment of the complex. Similar behaviour is also observed for the related complex [Os(TAP)2 (dppp2)]2+ upon quadruplex binding.

Time-resolved infra-red studies of photo-excited porphyrins in the presence of nucleic acids and in HeLa tumour cells: insights into binding site and electron transfer dynamics.

Cationic porphyrins based on the 5,10,15,20-meso-(tetrakis-4-N-methylpyridyl) core (TMPyP4) have been studied extensively over many years due to their strong interactions with a variety of nucleic acid structures, and their potential use as photodynamic therapeutic agents and telomerase inhibitors. In this paper, the interactions of metal-free TMPyP4 and Pt(II)TMPyP4 with guanine-containing nucleic acids are studied for the first time using time-resolved infrared spectroscopy (TRIR). In D2O solution (where the metal-free form exists as D2TMPyP4) both compounds yielded similar TRIR spectra (between 1450-1750 cm-1) following pulsed laser excitation in their Soret B-absorption bands. Density functional theory calculations reveal that vibrations centred on the methylpyridinium groups are responsible for the dominant feature at ca. 1640 cm-1. TRIR spectra of D2TMPyP4 or PtTMPyP4 in the presence of guanosine 5'-monophosphate (GMP), double-stranded {d(GC)5}2 or {d(CGCAAATTTGCG)}2 contain negative-going signals, 'bleaches', indicative of binding close to guanine. TRIR signals for D2TMPyP4 or PtTMPyP bound to the quadruplex-forming cMYC sequence {d(TAGGGAGGG)}2T indicate that binding occurs on the stacked guanines. For D2TMPyP4 bound to guanine-containing systems, the TRIR signal at ca. 1640 cm-1 decays on the picosecond timescale, consistent with electron transfer from guanine to the singlet excited state of D2TMPyP4, although IR marker bands for the reduced porphyrin/oxidised guanine were not observed. When PtTMPyP is incorporated into HeLa tumour cells, TRIR studies show protein binding with time-dependent ps/ns changes in the amide absorptions demonstrating TRIR's potential for studying light-activated molecular processes not only with nucleic acids in solution but also in biological cells.

Photophysical Properties and DNA Binding of Two Intercalating Osmium Polypyridyl Complexes Showing Light-Switch Effects.

The synthesis and photophysical characterization of two osmium(II) polypyridyl complexes, [Os(TAP)2dppz]2+ (1) and [Os(TAP)2dppp2]2+ (2) containing dppz (dipyrido[3,2-a:2',3'-c]phenazine) and dppp2 (pyrido[2',3':5,6]pyrazino[2,3-f][1,10]phenanthroline) intercalating ligands and TAP (1,4,5,8-tetraazaphenanthrene) ancillary ligands, are reported. The complexes exhibit complex electrochemistry with five distinct reductive redox couples, the first of which is assigned to a TAP-based process. The complexes emit in the near-IR (1 at 761 nm and 2 at 740 nm) with lifetimes of >35 ns with a low quantum yield of luminescence in aqueous solution (∼0.25%). The Δ and Λ enantiomers of 1 and 2 are found to bind to natural DNA and with AT and GC oligodeoxynucleotides with high affinities. In the presence of natural DNA, the visible absorption spectra are found to display significant hypochromic shifts, which is strongly evident for the ligand-centered π-π* dppp2 transition at 355 nm, which undergoes 46% hypochromism. The emission of both complexes increases upon DNA binding, which is observed to be sensitive to the Δ or Λ enantiomer and the DNA composition. A striking result is the sensitivity of Λ-2 to the presence of AT DNA, where a 6-fold enhancement of luminescence is observed and reflects the nature of the binding for the enantiomer and the protection from solution. Thermal denaturation studies show that both complexes are found to stabilize natural DNA. Finally, cellular studies show that the complexes are internalized by cultured mammalian cells and localize in the nucleus.

Hook, Line, and Sinker! Spectroscopic Studies of Bi-Modular Mono- and Bis-1,8-naphthalimide-Ru(bpy)3-conjugates as DNA "Light Switches".

Bi-chromophoric ruthenium polypyridyl complexes comprising one or two nitro-1,8-naphthalimide groups are shown to be effective DNA binders with off-on light switching properties. The binding to DNA was investigated using a combination of studies such as UV-visible absorption and emission titrations, thermal denaturation, and circular dichroism spectroscopy. The DNA affinity was shown to be sensitive to both the linker length and the number of naphthalimides (one vs two) contained in these systems and binding constants ranging from 106 to 107 M-1 for salmon testes DNA. The strong DNA binding is attributed to the combination of naphthalimide intercalation and the electrostatic interaction of the ruthenium complex. Large emission enhancements from the metal to ligand charge transfer (MLCT) emission arising from the metal complex were observed upon DNA binding, which was attributed to the interruption of intramolecular electron transfer quenching processes. Moving the nitro substitution from the 4-position to the 3-position is found to result in modification of the DNA binding and the resulting optical properties. The off-on light switch phenomena reported demonstrate the potential of these complexes to act as DNA probes.

Multiparametric nanoparticle-induced toxicity readouts with single cell resolution in HepG2 multicellular tumour spheroids.

The use of nanomaterials as therapeutic delivery vehicles requires their careful pre-clinical evaluation. Of particular importance in this regard is measurement of cellular toxicity, ideally assessing multiple parameters in parallel from various relevant subcellular organelles. In recent years it has become evident that in vitro monolayer-grown cells do not always accurately predict any toxicity response seen in vivo, and so there is a need for more sophisticated in vitro cell models, employing a greater depth of characterisation. In this work we present an automated high-content screening microscopy approach for quantifying nanoparticle-induced toxicity in a three-dimensional multicellular tumour spheroid (MCTS) cell model. As a proof-of-principle, we perform a comparative toxicity profile study of carboxylate- versus amine-modified polystyrene nanoparticles in HepG2 spheroids. Following treatment with these nanoparticle types, we demonstrate that several hundred spheroids, of various sizes, can be morphologically profiled in a single well using automated high-content image analysis. This provides a first level of information about spheroid health in response to nanoparticle treatment. Using a range of fluorescent reporters assessing membrane permeability, lysosome function and mitochondrial activity, we also show that nanoparticle-induced toxicity information can be obtained from individual cells with subcellular resolution. Strikingly, our work demonstrates that individual cells do not all behave in a consistent manner within a spheroid structure after exposure to nanoparticles. This highlights the need for toxicity studies to not only assess an appropriate number of spheroids, but also the importance of extracting information at the subcellular level.

Adenine Radical Cation Formation by a Ligand-Centered Excited State of an Intercalated Chromium Polypyridyl Complex Leads to Enhanced DNA Photo-oxidation.

Assessment of the DNA photo-oxidation and synthetic photocatalytic activity of chromium polypyridyl complexes is dominated by consideration of their long-lived metal-centered excited states. Here we report the participation of the excited states of [Cr(TMP)2dppz]3+ (1) (TMP = 3,4,7,8-tetramethyl-1,10-phenanthroline; dppz = dipyrido[3,2-a:2',3'-c]phenazine) in DNA photoreactions. The interactions of enantiomers of 1 with natural DNA or with oligodeoxynucleotides with varying AT content (0-100%) have been studied by steady state UV/visible absorption and luminescence spectroscopic methods, and the emission of 1 is found to be quenched in all systems. The time-resolved infrared (TRIR) and visible absorption spectra (TA) of 1 following excitation in the region between 350 to 400 nm reveal the presence of relatively long-lived dppz-centered states which eventually yield the emissive metal-centered state. The dppz-localized states are fully quenched when bound by GC base pairs and partially so in the presence of an AT base-pair system to generate purine radical cations. The sensitized formation of the adenine radical cation species (A•+T) is identified by assigning the TRIR spectra with help of DFT calculations. In natural DNA and oligodeoxynucleotides containing a mixture of AT and GC of base pairs, the observed time-resolved spectra are consistent with eventual photo-oxidation occurring predominantly at guanine through hole migration between base pairs. The combined targeting of purines leads to enhanced photo-oxidation of guanine. These results show that DNA photo-oxidation by the intercalated 1, which locates the dppz in contact with the target purines, is dominated by the LC centered excited state. This work has implications for future phototherapeutics and photocatalysis.

Porous Carbon Microparticles as Vehicles for the Intracellular Delivery of Molecules.

In this study the application of porous carbon microparticles for the transport of a sparingly soluble material into cells is demonstrated. Carbon offers an intrinsically sustainable platform material that can meet the multiple and complex requirements imposed by applications in biology and medicine. Porous carbon microparticles are attractive as they are easy to handle and manipulate and combine the chemical versatility and biocompatibility of carbon with a high surface area due to their highly porous structure. The uptake of fluorescently labeled microparticles by cancer (HeLa) and normal human embryonic Kidney (HEK 293) cells was monitored by confocal fluorescence microscopy. In this way the influence of particle size, surface functionalization and the presence of transfection agent on cellular uptake were studied. In the presence of transfection agent both large (690 nm) and small microparticles (250 nm) were readily internalized by both cell lines. However, in absence of the transfection agent the uptake was influenced by particle size and surface PEGylation with the smaller nanoparticle size being delivered. The ability of microparticles to deliver a fluorescein dye model cargo was also demonstrated in normal (HEK 293) cell line. Taken together, these results indicate the potential use of these materials as candidates for biological applications.

Synthesis, Characterization, and Biological Profiling of Ruthenium(II)-Based 4-Nitro- and 4-Amino-1,8-naphthalimide Conjugates.

We report the synthesis, photophysical characterization, and biological evaluation of four DNA-binding ruthenium(II) polypyridyl 4-nitro- and 4-amino-1,8-naphthalimide conjugates. A meta arrangement around the ring connecting the 1,8-naphthalimide to a bipyridine ligand creates a cleft, the result of which renders the shape of the complex complementary to that of DNA. We have demonstrated that each complex exhibits water solubility and a distinctive set of photophysical properties that has allowed the nature of their interaction with DNA to be probed by various ground- and excited-state titrations. Furthermore, by varying the ancillary ligands, we also demonstrate their ability to act as DNA photocleavers, where all compounds have been found to cleave supercoiled DNA with high efficiency. Detailed cellular uptake experiments revealed that the conjugates accumulate in the cytoplasm and nucleus of HeLa cells, showing characteristic red metal-to-ligand charge-transfer emission, and also exhibit photoactivated cytotoxicity within the cells upon irradiation at 450 nm. A comparison between the meta and para arrangements of the 1,8-naphthalimide moiety relative to the Ru(II) center suggests increased DNA binding in the case of the meta arrangement; however, bipyridine-4-amino-1,8-naphthalimide conjugates appear to show superior phototoxicity in comparison to their 4-nitro derivatives.

The influence of loops on the binding of the [Ru(phen)2dppz]2+ light-switch compound to i-motif DNA structures revealed by time-resolved spectroscopy.

Ultrafast time resolved infrared (TRIR) is used to report on the binding site of the "light-switch" complex [Ru(phen)2(dppz)]2+1 to i-motif structures in solution. Detailed information is provided due to perturbation of the local base vibrations by a 'Stark-like' effect which is used to establish the contribution of thymine base loop interactions to the binding site of 1 in this increasingly relevant DNA structure.

Caught in the Loop: Binding of the [Ru(phen)2 (dppz)]2+ Light-Switch Compound to Quadruplex DNA in Solution Informed by Time-Resolved Infrared Spectroscopy.

Ultrafast time-resolved infrared (TRIR) is used to report on the binding site of the [Ru(phen)2 (dppz)]2+ "light-switch" complex with both bimolecular (Oxytricha nova telomere) and intramolecular (human telomere) guanine-quadruplex structures in both K+ and Na+ containing solutions. TRIR permits the simultaneous monitoring both of the "dark" and "bright" states of the complex and of the quadruplex nucleobase bases, the latter via a Stark effect induced by the excited state of the complex. These data are used to establish the contribution of guanine base stacking and loop interactions to the binding site of this biologically relevant DNA structure in solution. A particularly striking observation is the strong thymine signal observed for the Na+ form of the human telomere sequence, which is expected to be in the anti-parallel conformation.

Understanding the factors controlling the photo-oxidation of natural DNA by enantiomerically pure intercalating ruthenium polypyridyl complexes through TA/TRIR studies with polydeoxynucleotides and mixed sequence oligodeoxynucleotides.

Ruthenium polypyridyl complexes which can sensitise the photo-oxidation of nucleic acids and other biological molecules show potential for photo-therapeutic applications. In this article a combination of transient visible absorption (TrA) and time-resolved infra-red (TRIR) spectroscopy are used to compare the photo-oxidation of guanine by the enantiomers of [Ru(TAP)2(dppz)]2+ in both polymeric {poly(dG-dC), poly(dA-dT) and natural DNA} and small mixed-sequence duplex-forming oligodeoxynucleotides. The products of electron transfer are readily monitored by the appearance of a characteristic TRIR band centred at ca. 1700 cm-1 for the guanine radical cation and a band centered at ca. 515 nm in the TrA for the reduced ruthenium complex. It is found that efficient electron transfer requires that the complex be intercalated at a G-C base-pair containing site. Significantly, changes in the nucleobase vibrations of the TRIR spectra induced by the bound excited state before electron transfer takes place are used to identify preferred intercalation sites in mixed-sequence oligodeoxynucleotides and natural DNA. Interestingly, with natural DNA, while it is found that quenching is inefficient in the picosecond range, a slower electron transfer process occurs, which is not found with the mixed-sequence duplex-forming oligodeoxynucleotides studied.

Investigation of the Cellular Destination of Fluorescently Labeled Carbon Nanohorns in Cultured Cells.

The high surface area, facile functionalization, and biocompatibility of carbon nanohorns (CNHs) make them attractive for many applications, including drug delivery. The cellular destination of nanomaterials dictates both the therapeutic application and the potential toxicity. Identifying the uptake mechanism is challenging as several endocytic pathways have been identified that facilitate cellular entry. Here, the cellular uptake of fluorescently labeled CNHs was assessed by utilizing quantitative cell-based assays to determine the factors influencing how internalization occurs and the destinations they reach in HeLa cells. Cell viability assays suggest that about 80% of the cells remained viable even at the highest concentration of 20 µg/mL exposure to CNHs. Uptake studies revealed that when pulse-chase conditions were applied, CNHs were seen to be localized both at the cell periphery and in a juxtanuclear pattern inside HeLa cells, in the latter case colocalizing with the lysosomal marker LAMP1. RNA interference studies, using a panel of RNA tools to individually deplete key molecules associated with the endocytic machinery, failed to block the internalization of CNHs into cells, suggesting that multiple mechanisms of endocytosis are used by this particle type.

Preparation of polymer gold nanoparticle composites with tunable plasmon coupling and their application as SERS substrates.

The controlled surface functionalisation of polystyrene beads (200 nm) with a lipoic acid derivative is used to assemble composites with between 4 to 20% loadings of citrate stabilised gold nanoparticles (13 nm-30 nm), which exhibit variable optical properties arising from interactions of the nanoparticle surface plasmon resonance (SPR). The decrease in average interparticle distance at higher loadings results in a red-shift in the SPR wavelength, which is well described by a universal ruler equation. The composite particles are shown to act as good SERS substrates for the standard analyte 4-mercaptophenol. The direct assessment of the SERS activity for individual composite particles solution is achieved by Raman optical tweezer measurements on 5.3 µm composite particles. These measurements show an increase in performance with increasing AuNP size. Importantly, the SERS activity of the individual particles compares well with the bulk measurements of samples deposited on a surface, indicating that the SERS activity arises primarily from the composite and not due to composite-composite interactions. In both studies the optimum SERS response is obtained with 30 nm AuNPs.

Carbon nanohorn modified platinum electrodes for improved immobilisation of enzyme in the design of glutamate biosensors.

Electrochemical enzymatic biosensors are the subject of research due to their potential for in vivo monitoring of glutamate, which is a key neurotransmitter whose concentration is related to healthy brain function. This study reports the use of biocompatible oxidised carbon nanohorns (o-CNH) with a high surface area, to enhance the immobilization of glutamate oxidase (GluOx) for improved biosensor performance. Two families of biosensors were designed to interact with the anionic GluOx. Family-1 consists of covalently functionalised o-CNH possessing hydrazide (HYZ) and amine (PEG-NH2) terminated surfaces and Family-2 comprised non-covalently functionalised o-CNH with different loadings of polyethyleneimine (PEI) to form a cationic hybrid. Amperometric detection of H2O2 formed by enzymatic oxidation of glutamate revealed a good performance from all designs with the most improved performance by the PEI hybrid systems. The best response was from a o-CNH : PEI ratio of 1 : 10 mg mL-1, which yielded a glutamate calibration plateau, JMAX, of 55 ± 9 µA cm-2 and sensitivity of 111 ± 34 µA mM-1 cm-2. The low KM of 0.31 ± 0.05 mM indicated the retention of the enzyme function, and a limit of detection of 0.02 ± 0.004 µM and a response time of 0.88 ± 0.13 s was determined. The results demonstrate the high sensitivity of these biosensors and their potential for future use for the detection of glutamate in vivo.

Template-Assisted Synthesis of Luminescent Carbon Nanofibers from Beverage-Related Precursors by Microwave Heating.

Luminescent carbon nanomaterials are important materials for sensing, imaging, and display technologies. This work describes the use of microwave heating for the template-assisted preparation of luminescent carbon nanofibers (CNFs) from the reaction of a range of beverage-related precursors with the nitrogen-rich polyethyleneimine. Highly luminescent robust carbon fibers that were 10 to 30 m in length and had a diameter of 200 nm were obtained under moderate conditions of temperature (250-260 °C) and a short reaction time (6 min). The high aspect ratio fibers showed wavelength-dependent emission that can be readily imaged using epifluorescence. The development of these multi-emissive one-dimensional (1D) carbon nanomaterials offers potential for a range of applications.

Spectro-electrochemical Studies on [Ru(TAP)2(dppz)]2+-Insights into the Mechanism of its Photosensitized Oxidation of Oligonucleotides.

[Ru(TAP)2(dppz)]2+ (TAP = 1,4,5,8-tetraazaphenanthrene; dppz = dipyrido[3,2- a:2',3'- c]phenazine) is known to photo-oxidize guanine in DNA. Whether this oxidation proceeds by direct photoelectron transfer or by proton-coupled electron transfer is still unknown. To help distinguish between these mechanisms, spectro-electrochemical experiments have been carried out with [Ru(TAP)2(dppz)]2+ in acetonitrile. The UV-vis and mid-IR spectra obtained for the one-electron reduced product were compared to those obtained by picosecond transient absorption and time-resolved infrared experiments of [Ru(TAP)2(dppz)]2+ bound to guanine-containing DNA. An interesting feature of the singly reduced species is an electronic transition in the near-IR region (with λmax at 1970 and 2820 nm). Density functional and time-dependent density functional theory simulations of the vibrational and electronic spectra of [Ru(TAP)2(dppz)]2+, the reduced complex [Ru(TAP)2(dppz)]+, and four isomers of [Ru(TAP)(TAPH)(dppz)]2+ (a possible product of proton-coupled electron transfer) were performed. Significantly, these predict absorption bands at λ > 1900 nm (attributed to a ligand-to-metal charge-transfer transition) for [Ru(TAP)2(dppz)]+ but not for [Ru(TAP)(TAPH)(dppz)]2+. Both the UV-vis and mid-IR difference absorption spectra of the electrochemically generated singly reduced species [Ru(TAP)2(dppz)]+ agree well with the transient absorption and time-resolved infrared spectra previously determined for the transient species formed by photoexcitation of [Ru(TAP)2(dppz)]2+ intercalated in guanine-containing DNA. This suggests that the photochemical process in DNA proceeds by photoelectron transfer and not by a proton-coupled electron transfer process involving formation of [Ru(TAP)(TAPH)(dppz)]2+, as is proposed for the reaction with 5'-guanosine monophosphate. Additional infrared spectro-electrochemical measurements and density functional calculations have also been carried out on the free TAP ligand. These show that the TAP radical anion in acetonitrile also exhibits strong broad near-IR electronic absorption (λmax at 1750 and 2360 nm).