Michele Iafisco

Surface enamel remineralization: biomimetic apatite nanocrystals and fluoride ions different effects

A new method for altered enamel surface remineralization has been proposed. To this aim carbonate-hydroxyapatite nanocrystals which mimic for composition, structure, nanodimensions, and morphology dentine apatite crystals and resemble closely natural apatite chemical-physical properties have been used The results underline the differences induced by the use of fluoride ions and hydroxyapatite nanocrystals in contrasting the mechanical abrasions and acid attacks to which tooth enamel is exposed. Fluoride ions generate a surface modification of the natural enamel apatite crystals increasing their crystallinity degree and relative mechanical and acid resistance. On the other hand, the remineralization produced by carbonate-hydroxyapatite consists in a deposition of a new apatitic mineral into the eroded enamel surface scratches. A new biomimetic mineral coating, which progressively fills and shadows surface scratches, covers and safeguards the enamel structure by contrasting the acid and bacteria attacks.

Adsorption and conformational change of myoglobin on biomimetic hydroxyapatite nanocrystals functionalized with alendronate.

The chemical conjugation of bisphosphonates (BPs), specifically alendronate, to hydroxyapatite could be an effective means to impart to it fine-tuned bioactivity. Horse heart myoglobin (Mb), a well-characterized protein, has been adsorbed onto biomimetic hydroxyapatite nanocrystals (nHA) and onto the nHA/alendronate conjugate powdered samples. The obtained materials have potential use in bone implantation and as prospective drug-delivery devices. The kinetic absorption of Mb onto nHA is dramatically affected by its functionalization with alendronate. The covering of the nHA surface by alendronate inhibits the adsorption of myoglobin. The adsorption mechanisms of the protein were studied by spectroscopic techniques (UV-vis and surface-enhanced Raman spectroscopy). The results indicate that the protein changes conformation upon adsorption on the inorganic substrate. In particular, the interaction with nHA alters the coordination state of the iron in the heme through the formation of a hexacoordinated low-spin Mb heme, possibly involving the distal histidine. Instead, the covering of the nHA surface by alendronate does not adsorb the protein but preserves the coordination state of the heme moiety. This study could be of significance either in the field of biomaterials science, in particular, to fine tune a bone-specific drug delivery device and to test nHA as a new support for heterogeneous catalysis, improving the understating of enzyme immobilization.

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.

Smart delivery of antitumoral platinum complexes from biomimetic hydroxyapatite nanocrystals

This study widens the role of biomimetic hydroxyapatite (HA) nanocrystals as bone substitutes and describes how they can be used as bone-specific drug delivery devices for in situ treatment of bone tumors upon local implantation. The adsorption and release kinetics of bis-{ethylenediamineplatinum(II)}-2-amino-1-hydroxyethane-1,1-diyl-bisphosphonate and bis-{ethylenediamineplatinum(II)}medronate on two kinds of HA nanocrystals having different morphologies, crystallinity degrees and surface areas have been investigated. The different chemical structures of the two Pt complexes appreciably affect not only the affinity towards the two kinds of HA, but also their release. The Pt complex loading is slightly greater for the HA characterized by lower crystallinity and higher surface area, with respect to the more crystalline one. The cytotoxicity of Pt complexes released from the HA were tested against human cervix carcinoma cells and, interestingly, were found to be more cytotoxic than the unmodified complexes. The released Pt species are therefore the active dichloridoethylenediamineplatinum(II) or related solvato species formed by Pt-bisphosphonate bond breaking.

The role of biomimetism in developing nanostructured inorganic matrices for drug delivery

Background: Biomimetism of synthetic biomaterials can be carried out at different levels, such as composition, structure, morphology, bulk and surface chemical–physical properties. Biomaterials can be turned into biomimetic imprinting of all these characteristics in order not only to optimise their interaction with biological tissues, but also to mimic biogenic materials in their functionalities. Objective: This review outlines the biomimetic chemical–physical properties of inorganic matrices in controlling drug release. Methods: This review is restricted to phosphates and silica among inorganic biomaterials proposed as drug delivery vehicles. Conclusion: By mimicking nature, we can design and synthesise inorganic smart materials that are reactive towards biological tissues and can release bioactive molecules by a kinetic that is controlled not only by the matrix tailored chemical–physical properties, but also by the response to stimuli induced by physiological or pathological processes.

Magnetic Bioinspired Hybrid Nanostructured Collagen–Hydroxyapatite Scaffolds Supporting Cell Proliferation and Tuning Regenerative Process

A bioinspired mineralization process was applied to develop biomimetic hybrid scaffolds made of (Fe(2+)/Fe(3+))-doped hydroxyapatite nanocrystals nucleated on self-assembling collagen fibers and endowed with super-paramagnetic properties, minimizing the formation of potentially cytotoxic magnetic phases such as magnetite or other iron oxide phases. Magnetic composites were prepared at different temperatures, and the effect of this parameter on the reaction yield in terms of mineralization degree, morphology, degradation, and magnetization was investigated. The influence of scaffold properties on cells was evaluated by seeding human osteoblast-like cells on magnetic and nonmagnetic materials, and differences in terms of viability, adhesion, and proliferation were studied. The synthesis temperature affects mainly the chemical-physical features of the mineral phase of the composites influencing the degradation, the microstructure, and the magnetization values of the entire scaffold and its biological performance. In vitro investigations indicated the biocompatibility of the materials and that the magnetization of the super-paramagnetic scaffolds, induced applying an external static magnetic field, improved cell proliferation in comparison to the nonmagnetic scaffold.

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.

Synthetic Biomimetic Carbonate-Hydroxyapatite Nanocrystals for Enamel Remineralization

New biomimetic carbonate-hydroxyapatite nanocrystals (CHA) have been designed and synthesized in order to obtain a remineralization of the altered enamel surfaces. Synthesized CHA mimic for composition, structure, nano dimension and morphology bone apatite crystals and their chemical-physical properties resemble closely those exhibited by enamel natural apatite. CHA can chemically bound themselves on the surface of natural enamel apatite thanks to their tailored biomimetic characteristics. The remineralization effect induced by CHA represents a real new deposition of carbonate-hydroxyapatite into the eroded enamel surface scratches forming a persistent biomimetic mineral coating, which covers and safeguards the enamel structure. The experimental results point out the possibility to use materials alternative to fluoride compounds which is commonly utilized to contrast the mechanical abrasions and acid attacks. The apatitic synthetic coating is less crystalline than enamel natural apatite, but consists of a new biomimetic apatitic mineral deposition which progressively fills the surface scratches. Therefore the application of biomimetic CHA may be considered an innovative approach to contrast the acid and bacteria attacks.

Conformational modifications of serum albumins adsorbed on different kinds of biomimetic hydroxyapatite nanocrystals

Nanosized carbonate-hydroxyapatite represents a suitable material for bone substitution and delivery of biomolecules. Its interaction with serum proteins plays a central role in the process of implantation of a device. Herein, surface interactions of human (HSA) and bovine (BSA) serum albumin with two biomimetic carbonate-hydroxyapatites (CHA) differing for size, surface area, crystallinity degree and surface properties have been investigated. BSA reached a different maximum coverage, being higher with the CHA nanocrystals with the higher surface area, the smaller size, the lower crystallinity degree and the plate shape morphology. On the contrary, HSA did not show significant differences of maximum coverage on the two substrates. The adsorption isotherms have been fitted by the Langmuir and Freundlich models, showing that the biomimetic hydroxyapatite matrix behaves as a mixture of energetically homogeneous and heterogeneous sites due to its surface disorder. By using Fourier Transform Infrared Spectroscopy (FT-IR) and Circular Dichroism (CD) it has been found that CHAs affect the conformation of the adsorbed proteins. FT-IR spectroscopy shows that protein adhesion on CHA surface results in a significant reduction in helicity for both proteins on both substrates, as function of the coating extent as well as modification in the beta-structures. CD spectroscopy of the HSA and BSA released in solution after desorption from the matrices shows that, while both proteins partially regain their helical structure, they show a distinct behaviour in their tertiary structure.

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.