Luminescent Citrate-Functionalized Terbium-Substituted Carbonated Apatite Nanomaterials: Structural Aspects, Sensitized Luminescence, Cytocompatibility, and Cell Uptake Imaging.

This work explores the preparation of luminescent and biomimetic Tb3+-doped citrate-functionalized carbonated apatite nanoparticles. These nanoparticles were synthesized employing a citrate-based thermal decomplexing precipitation method, testing a nominal Tb3+ doping concentration between 0.001 M to 0.020 M, and a maturation time from 4 h to 7 days. This approach allowed to prepare apatite nanoparticles as a single hydroxyapatite phase when the used Tb3+ concentrations were (i) ≤ 0.005 M at all maturation times or (ii) = 0.010 M with 4 h of maturation. At higher Tb3+ concentrations, amorphous TbPO4·nH2O formed at short maturation times, while materials consisting of a mixture of carbonated apatite prisms, TbPO4·H2O (rhabdophane) nanocrystals, and an amorphous phase formed at longer times. The Tb3+ content of the samples reached a maximum of 21.71 wt%. The relative luminescence intensity revealed an almost linear dependence with Tb3+ up to a maximum of 850 units. Neither pH, nor ionic strength, nor temperature significantly affected the luminescence properties. All precipitates were cytocompatible against A375, MCF7, and HeLa carcinogenic cells, and also against healthy fibroblast cells. Moreover, the luminescence properties of these nanoparticles allowed to visualize their intracellular cytoplasmic uptake at 12 h of treatment through flow cytometry and fluorescence confocal microscopy (green fluorescence) when incubated with A375 cells. This demonstrates for the first time the potential of these materials as nanophosphors for living cell imaging compatible with flow cytometry and fluorescence confocal microscopy without the need to introduce an additional fluorescence dye. Overall, our results demonstrated that Tb3+-doped citrate-functionalized apatite nanoparticles are excellent candidates for bioimaging applications.

Biomimetic Citrate-Coated Luminescent Apatite Nanoplatforms for Diclofenac Delivery in Inflammatory Environments.

Luminescent nanoparticles are innovative tools for medicine, allowing the imaging of cells and tissues, and, at the same time, carrying and releasing different types of molecules. We explored and compared the loading/release ability of diclofenac (COX-2 antagonist), in both undoped- and luminescent Terbium3+ (Tb3+)-doped citrate-coated carbonated apatite nanoparticles at different temperatures (25, 37, 40 °C) and pHs (7.4, 5.2). The cytocompatibility was evaluated on two osteosarcoma cell lines and primary human osteoblasts. Biological effects of diclofenac-loaded-nanoparticles were monitored in an in vitro osteoblast's cytokine-induced inflammation model by evaluating COX-2 mRNA expression and production of PGE2. Adsorption isotherms fitted the multilayer Langmuir-Freundlich model. The maximum adsorbed amounts at 37 °C were higher than at 25 °C, and particularly when using the Tb3+ -doped particles. Diclofenac-release efficiencies were higher at pH 5.2, a condition simulating a local inflammation. The luminescence properties of diclofenac-loaded Tb3+ -doped particles were affected by pH, being the relative luminescence intensity higher at pH 5.2 and the luminescence lifetime higher at pH 7.4, but not influenced either by the temperature or by the diclofenac-loaded amount. Both undoped and Tb3+-doped nanoparticles were cytocompatible. In addition, diclofenac release increased COX-2 mRNA expression and decreased PGE2 production in an in vitro inflammation model. These findings evidence the potential of these nanoparticles for osteo-localized delivery of anti-inflammatory drugs and the possibility to localize the inflammation, characterized by a decrease in pH, by changes in luminescence.

Crystallization, Luminescence and Cytocompatibility of Hexagonal Calcium Doped Terbium Phosphate Hydrate Nanoparticles.

Luminescent lanthanide-containing biocompatible nanosystems represent promising candidates as nanoplatforms for bioimaging applications. Herein, citrate-functionalized calcium-doped terbium phosphate hydrate nanophosphors of the rhabdophane type were prepared at different synthesis times and different Ca2+/Tb3+ ratios by a bioinspired crystallization method consisting of thermal decomplexing of Ca2+/Tb3+/citrate/phosphate/carbonate solutions. Nanoparticles were characterized by XRD, TEM, SEM, HR-TEM, FTIR, Raman, Thermogravimetry, inductively coupled plasma spectroscopy, thermoanalysis, dynamic light scattering, electrophoretic mobility, and fluorescence spectroscopy. They displayed ill-defined isometric morphologies with sizes ≤50 nm, hydration number n ~ 0.9, tailored Ca2+ content (0.42-8.11 wt%), and long luminescent lifetimes (800-2600 µs). Their relative luminescence intensities in solid state are neither affected by Ca2+, citrate content, nor by maturation time for Ca2+ doping concentration in solution below 0.07 M Ca2+. Only at this doping concentration does the maturation time strongly affect this property, decreasing it. In aqueous suspensions, neither pH nor ionic strength nor temperature affect their luminescence properties. All the nanoparticles displayed high cytocompatibility on two human carcinoma cell lines and cell viability correlated positively with the amount of doping Ca2+. Thus, these nanocrystals represent promising new luminescent nanoprobes for potential biomedical applications and, if coupled with targeting and therapeutic moieties, they could be effective tools for theranostics.

Self-Assembled Type I Collagen-Apatite Fibers with Varying Mineralization Extent and Luminescent Terbium Promote Osteogenic Differentiation of Mesenchymal Stem Cells.

This work explores in depth the simultaneous self-assembly and mineralization of type I collagen by a base-acid neutralization technique to prepare biomimetic collagen-apatite fibrils with varying mineralization extent and doped with luminescent bactericidal Tb3+ ions. Two variants of the method are tested: base-acid titration, a solution of Ca(OH)2 is added dropwise to a stirred solution containing type I collagen dispersed in H3 PO4 ; and direct mixing, the Ca(OH)2 solution is added by fast dripping onto the acidic solution. Only the direct mixing variant yielded an effective control of calcium phosphate polymorphism. Luminescence spectroscopy reveals the long luminescence lifetime and high relative luminescence intensity of the Tb3+ -doped materials, while two-photon confocal fluorescence microscopy shows the characteristic green fluorescence light when using excitation wavelength of 458 nm, which is not harmful to bone tissue. Cytotoxicity/viability tests reveal that direct mixing samples show higher cell proliferation than titration samples. Additionally, osteogenic differentiation essays show that all mineralized fibrils promote the osteogenic differentiation, but the effect is more pronounced when using samples prepared by direct mixing, and more notably when using the Tb3+ -doped mineralized fibrils. Based on these findings it is concluded that the new nanocomposite is an ideal candidate for bone regenerative therapy.

Bioinspired crystallization, sensitized luminescence and cytocompatibility of citrate-functionalized Ca-substituted europium phosphate monohydrate nanophosphors.

Biocompatible nanosystems exhibiting long-lifetime (∼millisecond) luminescence features are particularly relevant in the field of bioimaging. In this study, citrate-functionalized calcium-doped europium phosphates nanophosphors of the rhabdophane type were prepared at different synthesis times by a bioinspired crystallization route, consisting in thermal decomplexing of Ca2+/Eu3+ /citrate/phosphate/carbonate solutions. The general formula of this material is CaαEu1-α(PO4)1-α(HPO4)α·nH2O, with α ranging from 0 to 0.58 and n ∼ 1. A thorough characterization of the nanoparticles has been carried out by XRD (including data processing with Topas 6.0), HR-TEM, TEM, FTIR, TG/DTA, ICP, dynamic light scattering (DLS), electrophoretic mobility, and fluorescence spectroscopy. Based on these results a crystallization mechanism involving the filling of cationic sites with Ca2+ions associated to a concomitant adjustment of the PO4/HPO4 ratio was proposed. Upon calcium doping, the aspect ratio of the nanoparticles as well as of the crystalline domains decreased and the relative luminescence intensity (R.L.I.) could be modulated. Neither the pH nor the ionic strength, nor the temperature (from 25 to 37 °C) affected significantly the R.L.I. of particles after resuspension in water, leading to rather steady luminescence features usable in a large domain of conditions. This new class of luminescent compounds has been proved to be fully cytocompatible relative to GTL-16 human carcinoma cells and showed an improved cytocompatibility as the Ca2+ content increased when contacted with the more sensitive m17. ASC murine mesenchymal stem cells. These biocompatible nanoparticles thus appear as promising new tailorable tools for biomedical applications as luminescent nanoprobes.

Luminescent biomimetic citrate-coated europium-doped carbonated apatite nanoparticles for use in bioimaging: physico-chemistry and cytocompatibility.

Nanomedicine covers the application of nanotechnologies in medicine. Of particular interest is the setup of highly-cytocompatible nanoparticles for use as drug carriers and/or for medical imaging. In this context, luminescent nanoparticles are appealing nanodevices with great potential for imaging of tumor or other targetable cells, and several strategies are under investigation. Biomimetic apatite nanoparticles represent candidates of choice in nanomedicine due to their high intrinsic biocompatibility and to the highly accommodative properties of the apatite structure, allowing many ionic substitutions. In this work, the preparation of biomimetic (bone-like) citrate-coated carbonated apatite nanoparticles doped with europium ions is explored using the citrate-based thermal decomplexing approach. The technique allows the preparation of the single apatitic phase with nanosized dimensions only at Eu3+ doping concentrations ≤0.01 M at some timepoints. The presence of the citrate coating on the particle surface (as found in bone nanoapatites) and Eu3+ substituting Ca2+ is beneficial for the preparation of stable suspensions at physiological pH, as witnessed by the ζ-potential versus pH characterizations. The sensitized luminescence features of the solid particles, as a function of the Eu3+ doping concentrations and the maturation times, have been thoroughly investigated, while those of particles in suspensions have been investigated at different pHs, ionic strengths and temperatures. Their cytocompatibility is illustrated in vitro on two selected cell types, the GTL-16 human carcinoma cells and the m17.ASC murine mesenchymal stem cells. This contribution shows the potentiality of the thermal decomplexing method for the setup of luminescent biomimetic apatite nanoprobes with controlled features for use in bioimaging.

Direct observation of reversible electronic energy transfer involving an iridium center.

A cyclometalated iridium complex is reported where the core complex comprises naphthylpyridine as the main ligand and the ancillary 2,2'-bipyridine ligand is attached to a pyrene unit by a short alkyl bridge. To obtain the complex with satisfactory purity, it was necessary to modify the standard synthesis (direct reaction of the ancillary ligand with the chloro-bridged iridium dimer) to a method harnessing an intermediate tetramethylheptanolate-based complex, which was subjected to acid-promoted removal of the ancillary ligand and subsequent complexation. The photophysical behavior of the bichromophoric complex and a model complex without the pendant pyrene were studied using steady-state and time-resolved spectroscopies. Reversible electronic energy transfer (REET) is demonstrated, uniquely with an emissive cyclometalated iridium center and an adjacent organic chromophore. After excited-state equilibration is established (5 ns) as a result of REET, extremely long luminescence lifetimes of up to 225 µs result, compared to 8.3 µs for the model complex, without diminishing the emission quantum yield. As a result, remarkably high oxygen sensitivity is observed in both solution and polymeric matrices.

High performance optical sensing nanocomposites for low and ultra-low oxygen concentrations using phase-shift measurements.

The accurate and real-time measurement of low and ultra-low concentrations of oxygen using non-invasive methods is a necessity for a multitude of applications, from brewing beer to developing encapsulating barriers for optoelectronic devices. Current optical methods and sensing materials often lack the necessary sensitivity, signal intensity, or stability for practical applications. In this report we present a new optical sensing nanocomposite resulting in an outstanding overall performance when combined with the phase-shift measurement method (determination of luminescence lifetime in the frequency domain). For the first time we have incorporated the standard PtTFPP dye (PtTFPP = platinum(II) 5,10,15,20-meso-tetrakis-(2,3,4,5,6-pentafluorophenyl)-porphyrin) into AP200/19, a nanostructured aluminium oxide-hydroxide solid support. This sensing film shows an excellent sensitivity between 0 and 1% O2 (KSV = 3102 ± 132 bar⁻¹) and between 0 and 10% O2 (KSV = 2568 ± 614 bar⁻¹) as well as Δτ0.05% (62.53 ± 3.66%), which makes it 62 times more sensitive than PtTFPP immobilized in polystyrene and also 8 times more sensitive than PtTFPP immobilized on silica beads. Furthermore the phase-shift measurement method results in a significant improvement (about 23 times) in stability compared to the use of intensity recording methods. The film also displays full reversibility, long shelf stability (no change observed after 12 months), and it is not affected by humidity. To establish this sensing methodology and develop sensors over the full range of the visible light, we also studied three other dye-AP200/19 nanocomposites based on phosphorescent cyclometalated iridium(III) complexes.

Simple determination of the herbicide napropamide in water and soil samples by room temperature phosphorescence.

A new, simple, rapid and selective phosphorimetric method for determining napropamide is proposed which demonstrates the applicability of heavy-atom-induced room-temperature phosphorescence for analyzing pesticides in real samples. The phosphorescence signals are a consequence of intermolecular protection and are found exclusively with analytes in the presence of heavy atom salts. Sodium sulfite was used as an oxygen scavenger to minimize room-temperature phosphorescence quenching. The determination was performed in 1 M potassium iodide and 6 mM sodium sulfite at 20 degrees C. The phosphorescence intensity was measured at 520 nm with excitation at 290 nm. Phosphorescence was easily developed, with a linear relation to concentration between 3.2 and 600.0 ng ml(-1) and a detection limit of 3.2 ng ml(-1). The method has been successfully applied to the analysis of napropamide in water and soil samples and an exhaustive interference study was also carried out to display the selectivity of the proposed method.