José Luis Arias

Improving the brain delivery of gold nanoparticles by conjugation with an amphipathic peptide

BACKGROUND & AIMS Gold nanoparticles (GNPs) have promising applications for drug delivery as well as for the diagnosis and treatment of several pathologies, such as those related to the CNS. However, GNPs are retained in a number of organs, such as the liver and spleen. Owing to their negative charge and/or processes of opsonization, GNPs are retained by the reticuloendothelial system, thereby decreasing their delivery to the brain. It is therefore crucial to modify the nanoparticle surface in order to increase its lipophilicity and reduce its negative charge, thus achieving enhanced delivery to the brain. RESULTS In this article, we have shown that conjugation of 12 nm GNPs with the amphipathic peptide CLPFFD increases the in vivo penetration of these particles to the rat brain. The C(GNP)-LPFFD conjugates showed a smaller negative charge and a greater hydrophobic character than citrate-capped GNPs of the same size. We administered intraperitoneal injections of citrate GNPs and C(GNP)-LPFFD in rats, and determined the gold content in the tissues by neutron activation. Compared with citrate GNPs, the C(GNP)-LPFFD conjugate improved the delivery to the brain, increasing the concentration of gold by fourfold, while simultaneously reducing its retention by the spleen 1 and 2 h after injection. At 24 h, the conjugate was partially cleared from the brain, and mainly accumulated in the liver. The C(GNP)-LPFFD did not alter the integrity of the blood-brain barrier, and had no effect on cell viability.

Polysaccharides and proteoglycans in calcium carbonate-based biomineralization.

Biomineralization is a widespread phenomenon in nature leading to the formation of a variety of solid inorganic structures by living organisms, such as intracellular crystals in prokaryotes, exoskeletons in protozoa, algae, and invertebrates, spicules and lenses, bone, teeth, statoliths, and otoliths, eggshells, plant mineral structures, and also pathological biominerals such as gall stones, kidney stones, and oyster pearls. These biologically produced biominerals are inorganic-organic hybrid composites formed by self-assembled bottom up processes under mild conditions, showing interesting properties, controlled hierarchical structures, and remodeling or repair mechanisms which still remain to be developed into a practical engineering process. Therefore, the formation of biominerals provides a unique guide for the design of materials, especially those that need to be fabricated at ambient temperatures. In biominerals, the small amount of organic component not only reinforces the mechanical properties of the resulting composite but also exerts a crucial control on the mineralization process, contributing to the determination of the size, crystal morphology, specific crystallographic orientation, and superb properties of the particles formed. Therefore, biological routes of structuring biominerals are becoming valuable approaches for novel materials synthesis. Although several principles are applicable to the majority of the biominerals, herein we will focus on the role of polysaccharide polymers in calcium carbonate-based biominerals. As a general principle, the assembly of these biominerals consists of a four-stage process. It starts with the fabrication of a hydrophobic solid organic substrate or scaffolding onto which nucleation of the crystalline phase takes place closely associated with some polyanionic macromolecules. Crystal growth is then controlled by the addition of gel-structuring polyanionic macromolecules, and finally mineralization arrest is accompanied by the secretion of a new inert scaffolding of the same type or the deposition of other hydrophobic inhibitory macromolecules. Currently, a large number of proteins have been described which are involved in the control of biomineralization. These proteins are usually highly negatively charged and contain carboxylate, sulfate, or phosphate as functional groups, which may bind Ca ions and could control crystal nucleation and growth by lowering the interfacial energy between the crystal and the macromolecular substrate. However, the precise mechanism involved in controlling crystal nucleation, growth, and morphology is far from being understood. Combinatorial biology techniques have been recently developed for testing the ability of randomly generated peptides to bind different substrates or ions, thus allowing a correlation between peptide structure and ion binding affinity. However, the main focus is on the role of the backbone structure of the polymer due to the primary structure of the protein, because the synthetic technology does not allow the formation of post-translational modifications, such as sulfation and phosphorylation, which do occur in the eukaryotic cell. Even so, the occurrence of negatively charged groups in macromolecules involved in biomineralization, mainly derived from acidic amino acids, has inspired many researchers to produce synthetic polymers having such groups in order to control the size, orientation, phase, and morphology of inorganic crystals. However, since Abolins-Krogis’ work, a slow but increasing interest has been developed to explore the role of polysaccharides in biomineralization, despite the fact that their involvement in biomineralization seems to appear very early in evolution. There is no single type of polysaccharide associated with biominerals, but such polysaccharides are mainly hydroxylated, carboxylated, or sulfated or contain a mixture of these functional moieties.

Biomineralization and Eggshells: Cell-Mediated Acellular Compartments of Mineralized Extracellular Matrix

Publisher Summary This chapter summarizes cell biology, morphological organization, crystallography, chemical composition, process of mineralization, and biological function of avian eggshells. It also discusses emerging concepts of biomineralization and speculates about the mechanism of eggshell assembly and its implications for fabrication of polymer-ceramic composites. The eggshell is a microenvironmental compartment for housing developing embryos of a number of species. This unique microenvironment provides physical protection to the embryo and regulates gas, water, and ionic exchange. The avian eggshell can be characterized as a multilayered, polymer–ceramic composite. The three main layers include an outer mucous layer, an intermediate calcified zone, and an inner fibrous membrane layer. The shell membranes are the most internal layer of the eggshell and are formed by two nonmineralized fibrillar sublayers, the outer membrana testae externa, and the inner membrana testae interna or putaminis. The most external layer of the eggshell is referred to as the cuticle. This proteinaceous layer covers the entire calcified portion of the shell to a depth of about 10 μ m. The complementary use of biological, chemical, and crystallographic approaches demonstrates that the avian eggshell is a very promising model for the study of biomineralization. Avian eggshell is one of the most rapidly mineralizing biological systems known.

Collagens of the Chicken Eggshell Membranes

An immunohistochemical analysis of the eggshell membranes shows the occurrence of type X collagen while type I collagen was not detected by using an appropriate monoclonal antibody with untreated shell membranes. A positive immuno-reaction for type I collagen was obtained after digestion of the shell membranes with pepsin. These observations indicate the possibility that type I collagen epitope was masked by type X collagen and that type X collagen may serve as an inhibitory boundary for biomineralization.

Secretion pattern, ultrastructural localization and function of extracellular matrix molecules involved in eggshell formation.

The chicken eggshell is a composite bioceramic containing organic and inorganic phases. The organic phase contains, among other constituents, type X collagen and proteoglycans (mammillan, a keratan sulfate proteoglycan, and ovoglycan, a dermatan sulfate proteoglycan), whose localization depends on a topographically defined and temporally regulated deposition. Although the distribution of these macromolecules in the eggshell has been well established, little is known about their precise localization within eggshell substructures and oviduct cells or their pattern of production and function during eggshell formation. By using immunofluorescent and immuno-ultrastructural analyses, we examined the distribution of these macromolecules in oviduct cells at different post-oviposition times. To understand the role of proteoglycan sulfation on eggshell formation, we studied the effects of inhibition of proteoglycan sulfation by treatment with sodium chlorate. We showed that these macromolecules are produced by particular oviduct cell populations and at precise post-oviposition times. Based on the precise ultrastructural localization of these macromolecules in eggshell substructures, the timing of the secretion of these macromolecules by oviduct cells and the effects on eggshell formation caused by the inhibition of proteoglycan sulfation, the putative role of mammillan is in the nucleation of the first calcite crystals, while that of ovoglycan is to regulate the growth and orientation of the later forming crystals of the chicken eggshell.

Eggshells are shaped by a precise spatio-temporal arrangement of sequentially deposited macromolecules

The avian eggshell is a composite bioceramic which is formed by a controlled interaction of an organic and an inorganic phase. The organic phase contains, among other constituents, type X collagen and proteoglycans, mainly keratan and dermatan sulfate. Understanding the principles governing the synthesis and temporo-spatial distribution of such macromolecules, and their influence on the organization of the crystalline phase, is an essential aspect of establishing the biological basis of the quality of eggshell, both as an embryonic chamber and as a natural food package. In the present study, we have examined the process of eggshell formation by immunohistochemistry, scanning electron microscopy and energy dispersive X-ray microanalysis. Precise sites and timing of secretion were established for the deposition of particular macromolecules. Type X collagen is detected at the very first moment of shell membrane formation. The appearance of keratan sulfate coincides with the appearance of mammillae, while dermatan sulfate is deposited later, coincident with shell matrix deposition. We propose that keratan sulfate, due to its precise localization, temporal appearance and calcium-binding affinity, relates to the maintenance of calcium reserve bodies, the primary source of calcium for the embryo. On the other hand, dermatan sulfate may control crystal growth, resulting in a preferential orientation of calcite crystals within the palisade layer.

Squalene based nanocomposites: a new platform for the design of multifunctional pharmaceutical theragnostics.

This study reports the design of a novel theragnostic nanomedicine which combines (i) the ability to target a prodrug of gemcitabine to an experimental solid tumor under the influence of a magnetic field with (ii) the imaging of the targeted tumoral nodule. This concept is based on the inclusion of magnetite nanocrystals into nanoparticles (NPs) constructed by self-assembling molecules of the squalenoyl gemcitabine (SQgem) bioconjugate. The nanocomposites are characterized by an unusually high drug loading, a significant magnetic susceptibility, and a low burst release. When injected to the L1210 subcutaneous mice tumor model, these magnetite/SQgem NPs were magnetically guided, and they displayed considerably greater anticancer activity than the other anticancer treatments (magnetite/SQgem NPs nonmagnetically guided, SQgem NPs, or gemcitabine free in solution). The histology and immunohistochemistry investigation of the tumor biopsies clearly evidenced the therapeutic superiority of the magnetically guided nanocomposites, while Prussian blue staining confirmed their accumulation at the tumor periphery. The superior therapeutic activity and enhanced tumor accumulation has been successfully visualized using T(2)-weighted imaging in magnetic resonance imaging (MRI). This concept was further enlarged by (i) the design of squalene-based NPs containing the T(1) Gd(3+) contrast agent instead of magnetite and (ii) the application to other anticancer squalenoyls, such as, cisplatin, doxorubicin, and paclitaxel. Thus, by combining different anticancer medicines as well as contrast imaging agents in NPs, we open the door toward generic conceptual framework for cancer treatment and diagnosis. This new theragnostic nanotechnology platform is expected to have important applications in cancer therapy.

Role of type X collagen on experimental mineralization of eggshell membranes.

Type X collagen is a transient and developmentally regulated collagen that has been postulated to be involved in controlling the later stages of endochondral bone formation. However, the role of this collagen in these events is not yet known. In order to understand the function of type X collagen, if any, in the process of biomineralization, the properties of type X collagen in eggshell membranes were further investigated. Specifically, calvaria-derived osteogenic cells were tested for their ability to mineralize eggshell membranes in vitro. Immunohistochemistry with specific monoclonal antibodies was used to correlate the presence or absence of type X collagen or its propeptide domains with the ability of shell membranes to be mineralized. The extent of mineralization was assessed by Von Kossa staining, scanning electron microscopy and energy-dispersive spectroscopy. The results indicate that the non-helical domains of type X collagen must be removed to facilitate the cell-mediated mineralization of eggshell membranes. In this tissue, intact type X collagen does not appear to stimulate or support cell-mediated mineralization. We postulate that the non-helical domains of type X collagen function in vivo to inhibit mineralization and thereby establish boundaries which are protected from mineral deposition.

Magnetoresponsive squalenoyl gemcitabine composite nanoparticles for cancer active targeting.

Gemcitabine is widely used against a variety of solid tumors; however, it possesses some important drawbacks such as rapid deamination leading to short biological half-life and induction of tumor resistance. We have shown previously that the covalent coupling of squalene (a precursor of cholesterol in sterol biosynthesis) to gemcitabine resulted in a potent nanomedicine, squalenoyl gemcitabine (SQdFdC), which displayed appreciable anticancer activity. Now, the present study describes the concept of magnetic responsiveness of SQdFdC nanoparticles obtained by the nanoprecipitation of SQdFdC around magnetite nanoparticles. To investigate these new core/shell nanoparticles, we have compared their structure, chemical composition and surface properties with those of either the magnetic core alone or of the SQdFdC coating material. X-ray diffraction and infrared spectroscopy studies have shown that the composite core/shell particles displayed an intermediate behavior between that of pure magnetite and of pure SQdFdC nanoparticles, whereas dark-field, high-resolution transmission electron microscopy allowed clear demonstration of the core/shell structure. Electrophoresis measurements as a function of both pH and ionic strength, as well as thermodynamic consideration, showed similar behavior of core/shell and pure SQdFdC nanoparticles, suggesting again the coating of the magnetite core by the SQdFdC prodrug. The two important parameters to be controlled in the efficient adsorption of SQdFdC onto magnetite nanocores were the magnetite/SQdFdC weight ratio and the pluronic F-68 concentration. Pluronic F-68 was found to play a key role as a surfactant in the generation of stable composite core/shell nanoparticle suspensions. Finally, the characterization of the magnetic properties of these core/shell nanoparticles revealed that if the squalenoyl shell reduced the magnetic responsiveness of the particles, it kept unchanged their soft ferrimagnetic character. Thus, the heterogeneous structure of these nanoparticles could confer them both magnetic field responsiveness and potential applicability as a drug carrier for active targeting to solid tumors.

Sulfated polymers in biological mineralization: a plausible source for bio-inspired engineering

Biomineralization leads to the formation of inorganic crystals with unique, ordered, refined shapes that are regulated by specific macromolecules. This process has been a source of inspiration for exploring novel approaches to the fabrication of inorganic-based surfaces and interfaces. Among those macromolecules, sulfated polymers, referred to as proteoglycans, have not received enough attention, although there is increasing evidence of their widespread occurrence in biominerals. Here we examine the available information on the nature, distribution and possible role of sulfated polymers in biomineralization, and highlight new directions to stimulate further research activities.