Hydrothermal Transformation of Eggshell Calcium Carbonate into Apatite Micro-Nanoparticles: Cytocompatibility and Osteoinductive Properties.

The eggshell is a biomineral consisting of CaCO3 in the form of calcite phase and a pervading organic matrix (1-3.5 wt.%). Transforming eggshell calcite particles into calcium phosphate (apatite) micro-nanoparticles opens the door to repurposing the eggshell waste as materials with potential biomedical applications, fulfilling the principles of the circular economy. Previous methods to obtain these particles consisted mainly of two steps, the first one involving the calcination of the eggshell. In this research, direct transformation by a one-pot hydrothermal method ranging from 100-200 °C was studied, using suspensions with a stoichiometric P/CaCO3 ratio, K2HPO4 as P reagent, and eggshells particles (Ø < 50 µm) both untreated and treated with NaClO to remove surface organic matter. In the untreated group, the complete conversion was achieved at 160 °C, and most particles displayed a hexagonal plate morphology, eventually with a central hole. In the treated group, this replacement occurred at 180 °C, yielding granular (spherulitic) apatite nanoparticles. The eggshell particles and apatite micro-nanoparticles were cytocompatible when incubated with MG-63 human osteosarcoma cells and m17.ASC murine mesenchymal stem cells and promoted the osteogenic differentiation of m17.ASC cells. The study results are useful for designing and fabricating biocompatible microstructured materials with osteoinductive properties for applications in bone tissue engineering and dentistry.

A sustainable one-pot method to transform seashell waste calcium carbonate to osteoinductive hydroxyapatite micro-nanoparticles.

We have developed a straightforward, one-pot, low-temperature hydrothermal method to transform oyster shell waste particles (bCCP) from the species Crassostrea gigas (Mg-calcite, 5 wt% Mg) into hydroxyapatite (HA) micro/nanoparticles. The influence of the P reagents (H3PO4, KH2PO4, and K2HPO4), P/bCCP molar ratios (0.24, 0.6, and 0.96), digestion temperatures (25-200 °C), and digestion times (1 week-2 months) on the transformation process was thoroughly investigated. At 1 week, the minimum temperature to yield the full transformation significantly reduced from 160 °C to 120 °C when using K2HPO4 instead of KH2PO4 at a P/bCCP ratio of 0.6, and even to 80 °C at a P/bCCP ratio of 0.96. The transformation took place via a dissolution-reprecipitation mechanism driven by the favorable balance between HA precipitation and bCCP dissolution, due to the lower solubility product of HA than that of calcite at any of the tested temperatures. Both the bCCP and the derived HA particles were cytocompatible for MG-63 human osteosarcoma cells and m17.ASC murine mesenchymal stem cells, and additionally, they promoted the osteogenic differentiation of m17.ASC, especially the HA particles. Because of their physicochemical features and biological compatibility, both particles could be useful osteoinductive platforms for translational applications in bone tissue engineering.

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.

Production of Cross-Linked Lipase Crystals at a Preparative Scale.

The autoimmobilization of enzymes via cross-linked enzyme crystals (CLECs) has regained interest in recent years, boosted by the extensive knowledge gained in protein crystallization, the decrease of cost and laboriousness of the process, and the development of potential applications. In this work, we present the crystallization and preparative-scale production of reinforced cross-linked lipase crystals (RCLLCs) using a commercial detergent additive as a raw material. Bulk crystallization was carried out in 500 mL of agarose media using the batch technique. Agarose facilitates the homogeneous production of crystals, their cross-linking treatment, and their extraction. RCLLCs were active in an aqueous solution and in hexane, as shown by the hydrolysis of p-nitrophenol butyrate and α-methylbenzyl acetate, respectively. RCLLCs presented both high thermal and robust operational stability, allowing the preparation of a packed-bed chromatographic column to work in a continuous flow. Finally, we determined the three-dimensional (3D) models of this commercial lipase crystallized with and without phosphate at 2.0 and 1.7 Å resolutions, respectively.

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.

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.