José Manuel Delgado-López

Crystallization of bioinspired citrate-functionalized nanoapatite with tailored carbonate content.

Novel citrate-functionalized carbonate-apatite nanoparticles with mean lengths ranging from 20 to 100 nm were synthesized by a thermal-decomplexing batch method. Needle-like and plate-shaped morphologies were obtained in the absence and presence of sodium carbonate in the precipitation medium, respectively. The precipitation time and the presence of sodium carbonate strongly affect the chemical composition as well as the dimensions and the crystallinity of nanoparticles. At a short precipitation time, poorly crystalline apatites of 100 nm mean length with a low degree of carbonation (1.5% w/w, mainly in B-position) and a high citrate content (5.9% w/w) were precipitated. This citrate content is close to that recently measured in bone apatite. When increasing the precipitation time up to 96 h the mean length and the citrate content progressively decrease and at the same time the nanoparticles become more crystalline. They are composed of a well-ordered carbonate-substituted apatitic core embedded in a non-apatitic hydrated layer containing citrate ions. This layer progressively transforms into a more stable apatite domain upon maturation in aqueous media. The nanoparticles displayed excellent compatibility properties in cell biological systems, since they were not cytotoxic to a mouse carcinoma cell line when added to a final concentration of 100 μgml(-1). This work provides new insights into the role of citrate on the crystallization of nanoapatites. Moreover, the synthesized nanoparticles are promising materials for use as nanocarriers for local targeted drug delivery systems as well as building blocks for the preparation of nanostructured scaffolds for cells in bone tissue engineering.

PH-responsive delivery of doxorubicin from citrate-apatite nanocrystals with tailored carbonate content

In this work, the efficiency of bioinspired citrate-functionalized nanocrystalline apatites as nanocarriers for delivery of doxorubicin (DOXO) has been assessed. The nanoparticles were synthesized by thermal decomplexing of metastable calcium/citrate/phosphate solutions both in the absence (Ap) and in the presence (cAp) of carbonate ions. The presence of citrate and carbonate ions in the solution allowed us to tailor the size, shape, carbonate content, and surface chemistry of the nanoparticles. The drug-loading efficiency of the two types of apatite was evaluated by means of the adsorption isotherms, which were found to fit a Langmuir-Freundlich behavior. A model describing the interaction between apatite surface and DOXO is proposed from adsorption isotherms and ζ-potential measurements. DOXO is adsorbed as a dimer by means of a positively charged amino group that electrostatically interacts with negatively charged surface groups of nanoparticles. The drug-release profiles were explored at pHs 7.4 and 5.0, mimicking the physiological pH in the blood circulation and the more acidic pH in the endosome-lysosome intracellular compartment, respectively. After 7 days at pH 7.4, cAp-DOXO released around 42% less drug than Ap-DOXO. However, at acidic pH, both nanoassemblies released similar amounts of DOXO. In vitro assays analyzed by confocal microscopy showed that both drug-loaded apatites were internalized within GTL-16 human carcinoma cells and could release DOXO, which accumulated in the nucleus in short times and exerted cytotoxic activity with the same efficiency. cAp are thus expected to be a more promising nanocarrier for experiments in vivo, in situations where intravenous injection of nanoparticles are required to reach the targeted tumor, after circulating in the bloodstream.

Cell Surface Receptor Targeted Biomimetic Apatite Nanocrystals for Cancer Therapy

Nanosized drug carriers functionalized with moieties specifically targeting tumor cells are promising tools in cancer therapy, due to their ability to circulate in the bloodstream for longer periods and their selectivity for tumor cells, enabling the sparing of healthy tissues. Because of its biocompatibility, high bioresorbability, and responsiveness to pH changes, synthetic biomimetic nanocrystalline apatites are used as nanocarriers to produce multifunctional nanoparticles, by coupling them with the chemotherapeutic drug doxorubicin (DOXO) and the DO-24 monoclonal antibody (mAb) directed against the Met/Hepatocyte Growth Factor receptor (Met/HGFR), which is over-expressed on different types of carcinomas and thus represents a useful tumor target. The chemical-physical features of the nanoparticles are fully investigated and their interaction with cells expressing (GTL-16 gastric carcinoma line) or not expressing (NIH-3T3 fibroblasts) the Met/HGFR is analyzed. Functionalized nanoparticles specifically bind to and are internalized in cells expressing the receptor (GTL-16) but not in the ones that do not express it (NIH-3T3). Moreover they discharge DOXO in the targeted GTL-16 cells that reach the nucleus and display cytotoxicity as assessed in an MTT assay. Two different types of ternary nanoparticles are prepared, differing for the sequence of the functionalization steps (adsorption of DOXO first and then mAb or vice versa), and it is found that the ones in which mAb is adsorbed first are more efficient under all the examined aspects (binding, internalization, cytotoxicity), possibly because of a better mAb orientation on the nanoparticle surface. These multifunctional nanoparticles could thus be useful instruments for targeted local or systemic drug delivery, allowing a reduction in the therapeutic dose of the drug and thus adverse side effects. Moreover, this work opens new perspectives in the use of nanocrystalline apatites as a new platform for theranostic applications in nanomedicine.

Evolution of calcium phosphate precipitation in hanging drop vapor diffusion by in situ Raman microspectroscopy

The time-evolution of calcium phosphate precipitation by vapor diffusion has been studied by in situ confocal Raman microspectroscopy. A hanging drop configuration within a device known as “crystallization mushroom” was employed in order to improve the Raman signal coming from growing crystals. This innovative methodology allowed to identify and follow the evolution of the precipitates formed at different areas of the drops containing mixed solutions of Ca(CH3COO)2 and (NH4)2HPO4 due to the diffusion of CO2 and NH3 gases released from NH4HCO3 solutions at different concentrations (30 mM, 100 mM and 2 M). Time-dependent in situ Raman spectra indicated that amorphous calcium phosphate (ACP) was the first precipitate appearing just after mixing the Ca- and PO4-containing solutions. A few minutes later, it transformed to dicalcium phosphate dihydrate (DCPD). The lifetime of DCPD strongly depends on the concentration of the NH4HCO3 solutions and thus on the pH increase rate. The pathway for the phase transformation from ACP to DCPD and then to octacalcium phosphate (OCP) followed a dissolution–reprecipitation mechanism. Additionally, OCP acted as temporal template for the heterogeneous nucleation and crystallization of biomimetic carbonate–apatite nanocrystals (cAp). The characterization by TEM, XRPD and Raman spectroscopy of the freeze-dried powders obtained after seven days confirmed that OCP and cAp were the remaining phases when using 30 mM and 100 mM NH4HCO3 solutions. By contrast, working with the highest NH4HCO3 concentration the system evolved to the precipitation of elongated calcite crystals.

Crystallization of citrate-stabilized amorphous calcium phosphate to nanocrystalline apatite: a surface-mediated transformation

This work explores the mechanisms underlying the crystallization of citrate-functionalized amorphous calcium phosphate (cit-ACP) in two relevant media, combining in situ and ex situ characterization techniques. Results demonstrate that citrate desorption from cit-ACP triggers the surface-mediated transformation to nanocrystalline apatite (Ap). Our findings shed light on the key role of citrate, an important component of bone organic matrix, and the medium composition in controlling the rate of transformation and the morphology of the resulting Ap phase.

Biomimetic mineralization of recombinant collagen type I derived protein to obtain hybrid matrices for bone regeneration

Understanding the mineralization mechanism of synthetic protein has recently aroused great interest especially in the development of advanced materials for bone regeneration. Herein, we propose the synthesis of composite materials through the mineralization of a recombinant collagen type I derived protein (RCP) enriched with RGD sequences in the presence of magnesium ions (Mg) to closer mimic bone composition. The role of both RCP and Mg ions in controlling the precipitation of the mineral phase is in depth evaluated. TEM and X-ray powder diffraction reveal the crystallization of nanocrystalline apatite (Ap) in all the evaluated conditions. However, Raman spectra point out also the precipitation of amorphous calcium phosphate (ACP). This amorphous phase is more evident when RCP and Mg are at work, indicating the synergistic role of both in stabilizing the amorphous precursor. In addition, hybrid matrices are prepared to tentatively address their effectiveness as scaffolds for bone tissue engineering. SEM and AFM imaging show an homogeneous mineral distribution on the RCP matrix mineralized in presence of Mg, which provides a surface roughness similar to that found in bone. Preliminary in vitro tests with pre-osteoblast cell line show good cell-material interaction on the matrices prepared in the presence of Mg. To the best of our knowledge this work represents the first attempt to mineralize recombinant collagen type I derived protein proving the simultaneous effect of the organic phase (RCP) and Mg on ACP stabilization. This study opens the possibility to engineer, through biomineralization process, advanced hybrid matrices for bone regeneration.

Monoclonal antibody-targeted fluorescein-5-isothiocyanate-labeled biomimetic nanoapatites: a promising fluorescent probe for imaging applications.

Multifunctional biomimetic nanoparticles (NPs) are acquiring increasing interest as carriers in medicine and basic research since they can efficiently combine labels for subsequent tracking, moieties for specific cell targeting, and bioactive molecules, e.g., drugs. In particular, because of their easy synthesis, low cost, good biocompatibility, high resorbability, easy surface functionalization, and pH-dependent solubility, nanocrystalline apatites are promising candidates as nanocarriers. This work describes the synthesis and characterization of bioinspired apatite nanoparticles to be used as fluorescent nanocarriers targeted against the Met/hepatocyte growth factor receptor, which is considered a tumor associated cell surface marker of many cancers. To this aim the nanoparticles have been labeled with Fluorescein-5-isothiocyanate (FITC) by simple isothermal adsorption, in the absence of organic, possibly toxic, molecules, and then functionalized with a monoclonal antibody (mAb) directed against such a receptor. Direct labeling of the nanoparticles allowed tracking the moieties with spatiotemporal resolution and thus following their interaction with cells, expressing or not the targeted receptor, as well as their fate in vitro. Cytofluorometry and confocal microscopy experiments showed that the functionalized nanocarriers, which emitted a strong fluorescent signal, were rapidly and specifically internalized in cells expressing the receptor. Indeed, we found that, once inside the cells expressing the receptor, mAb-functionalized FITC nanoparticles partially dissociated in their two components, with some mAbs being recycled to the cell surface and the FITC-labeled nanoparticles remaining in the cytosol. This work thus shows that FITC-labeled nanoapatites are very promising probes for targeted cell imaging applications.

pH-responsive collagen fibrillogenesis in confined droplets induced by vapour diffusion.

A novel methodology for the assembly of collagen fibrils in microliter drops is proposed. It consists in the gradual increase of pH by means of vapour diffusion coming from the decomposition of NH4HCO3 solutions. The pH increase rate as well as the final steady pH of solutions containing collagen can be adjusted by varying the concentration of NH4HCO3. Both parameters are of predominant importance in collagen fibrillogenesis. The effect of these parameters on the kinetic of the fibrillogenesis process and on the fibrils morphology was studied. We found that both the kinetic and the morphology are mainly driven by electrostatic interactions. A gradual increase of pH slows down the formation of collagen fibres and favours the lateral interaction between fibrils producing broader fibres. On the other hand, a rapid increase of pH reduces the lateral electrostatic interactions favouring the formation of thinner fibres. The formation of the D-band periodicity is also a pH-dependent process that occurs after fibrillogenesis when the most stable state of fibres formation has been reached.

Control Over Nanocrystalline Apatite Formation: What Can the X-Ray Total Scattering Approach Tell Us

Living organisms are able to induce and control the nucleation and crystallization of a wide variety of minerals. Vertebrates use calcium phosphates to build their mineral phases in hard tissues (i.e. bone, dentin and tooth enamel) and in pathological deposits (e.g. dental and urinary calculus and stones, atherosclerotic lesions). Understanding how organisms form their extremely specialized mineralized structures and the in vivo mechanisms enabling their control over crystal morphology, size and polymorphism and, ultimately, over functional properties is particularly important. Nevertheless, these systems are usually complex hybrids very difficult to be fully characterized. Bone is one of the most studied mineralized tissues, although many important aspects of its sophisticated mineralization process need further investigations. In this chapter, we highlight the role of citrate in driving the formation of platy-shaped bio-inspired apatite. Recent solid-state nuclear magnetic resonance (NMR) studies evidenced that citrate, which accounts for ~5.5 % wt of the total organic component of bone, is strongly bound to bone apatite platelets. Thus, we also discuss the possible role of citrate in inducing the typical platelike morphology of bone apatite. Furthermore, the chapter aims at highlighting the strength of X-ray total scattering for characterizing nanocrystalline apatites in terms of crystal structure and defects, stoichiometry (i.e. Ca/P ratio), size and morphology. Future perspectives on the use of time-resolved experiments and combination of complementary advanced techniques are briefly outlined aiming to enhance the fundamental knowledge of bone biomineralization at atomic and nanometre length scales.

Crystallization of monohydrocalcite in a silica-rich alkaline solution

Monohydrocalcite was crystallized in a silica-rich alkaline solution at room temperature using the counter diffusion method (CDM) in the absence of magnesium, by the reaction of calcium chloride and sodium carbonate. Field emission scanning electron microscopy observations showed monohydrocalcite crystals that were hundreds of micrometers in size exhibiting a unique multi-layered structure. X-ray diffraction and Raman microspectroscopy characterizations demonstrated that monohydrocalcite remained in a stable phase for a number of months. After monitoring the crystallization process by in situ Raman microspectroscopy, it was found that monohydrocalcite was the initial phase, with no phase transformation occurring during the crystal growth. This work demonstrates that silica plays a key role in the formation and stabilization of the monohydrocalcite phase.