Nikolaos Bouropoulos

Calcium Oxalate Crystals in Tomato and Tobacco Plants: Morphology and in Vitro Interactions of Crystal‐Associated Macromolecules

Plants form calcium oxalate crystals with unique morphologies under well-controlled conditions. We studied the morphology of single calcium oxalate monohydrate (whewellite) crystals extracted from tomato and tobacco leaves. These crystals have a pseudotetrahedral shape. We identified the (101), (101) or (102), (110), and (hk0) faces as stable faces. The morphology is chiral with unique handedness. We also show that calcium oxalate monohydrate crystals isolated from tomato, tobacco, and bougainvillea leaves contain macromolecules rich in Gly, Glx, and Ser. Crystal-associated macromolecules extracted from tomato and tobacco influence the morphology of calcium oxalate monohydrate crystals grown in vitro, promoting preferential development of the [120] faces. Furthermore, crystal-associated macromolecules from tobacco promote nucleation of calcium oxalate monohydrate crystals, whereas model polypeptides do not have any significant effect on nucleation. These results imply an active role of the crystal-associated macromolecules in the formation of pseudotetrahedral shapes in vitro, and these properties may in part be responsible for the unique chiral morphology of the natural pyramidal-shaped crystals.

Thermosensitive hydrogels for nasal drug delivery: The formulation and characterisation of systems based on N-trimethyl chitosan chloride

Towards the development of a thermosensitive drug-delivery vehicle for nasal delivery, a systematic series of N-trimethyl chitosan chloride polymers, synthesised from chitosans of three different average molecular weights, have been co-formulated into a hydrogel with poly(ethylene glycol) and glycerophosphate. Rheological evaluations have shown that hydrogels derived from N-trimethyl chitosan with a low degree of quaternisation and high or medium average molecular weight exhibit relatively short sol-gel transition times at physiologically relevant temperatures. Also, the same hydrogels display good water-holding capacity and strong mucoadhesive potential, and their mixtures with mucus exhibit rheological synergy. An aqueous hydrogel formulation, derived from N-trimethyl chitosan of medium average molecular weight and low degree of quaternisation, appears particularly promising in that it exhibits most favourable rheological and mucoadhesive behaviour and a sol-gel transition that occurs at 32.5°C within 7 min.

Electrospun PVP-indomethacin constituents for transdermal dressings and drug delivery devices.

A method in layering dressings with a superficial active layer of sub-micrometer scaled fibrous structures is demonstrated. For this, polyvinylpyrolidone (PVP)-indomethacin (INDO) fibres (5% w/v PVP, 5% w/w indomethacin, using a 50:50 ethanol-methanol solvent system) were produced at different flow rates (50 μL/min and 100 μL/min) via a modified electrospinning device head (applied voltage varied between 15 ± 2 kV). We further assessed these structures for their morphological, physical and chemical properties using SEM, AFM, DSC, XRD, FTIR and HPLC-UV. The average diameter of the resulting 3D (ca. 500 nm in height) PVP-INDO fibres produced at 50 μL/min flow rate was 2.58 ± 0.30 μm, while an almost two-fold increase in the diameter was observed (5.22 ± 0.83 μm) when the flow rate was doubled. However, both of these diameters were appreciably smaller than the existing dressing fibres (ca. 30 μm), which were visible even when layered with the active spun fibres. Indomethacin was incorporated in the amorphous state. The encapsulation efficiency was 75% w/w, with complete drug release in 45 min. The advantages are the ease of fabrication and deposition onto any existing normal or functionalised dressing (retaining the original fabric functionality), elimination of topical product issues (application, storage and transport), rapid release of active and controlled loading of drug content (fibre layer).

Pore Geometry Regulates Early Stage Human Bone Marrow Cell Tissue Formation and Organisation

Porous architecture has a dramatic effect on tissue formation in porous biomaterials used in regenerative medicine. However, the wide variety of 3D structures used indicates there is a clear need for the optimal design of pore architecture to maximize tissue formation and ingrowth. Thus, the aim of this study was to characterize initial tissue growth solely as a function of pore geometry. We used an in vitro system with well-defined open pore slots of varying width, providing a 3D environment for neo-tissue formation while minimizing nutrient limitations. Results demonstrated that initial tissue formation was strongly influenced by pore geometry. Both velocity of tissue invasion and area of tissue formed increased as pores became narrower. This is associated with distinct patterns of actin organisation and alignment depending on pore width, indicating the role of active cell generated forces. A mathematical model based on curvature driven growth successfully predicted both shape of invasion front and constant rate of growth, which increased for narrower pores as seen in experiments. Our results provide further evidence for a front based, curvature driven growth mechanism depending on pore geometry and tissue organisation, which could provide important clues for 3D scaffold design.

Controlled release of 5-fluorouracil from microporous zeolites.

UNLABELLED Zeolite particles with different pore diameter and particle size were loaded with the model anticancer drug 5-fluorouracil. The loaded zeolites were characterized by means of SEM, XRD, DSC, XPS, N2 physisorption and FT-IR. Higher loading of 5-FU was observed for NaX-FAU than BEA. Release studies were carried out in HCl 0.1N. Release of 5-FU from NaX-FAU showed exponential-type behaviour with the drug fully released within 10 min. In the case of BEA, the kinetics of 5-FU shows a multi-step profile with prolonged release over time. Molecular dynamics simulations showed that diffusion of the drug molecule through the BEA framework is lower than for NaX-FAU due to increased van der Waals interaction between the drug and the framework. The effect of zeolitic particles on the viability of Caco-2 monolayers showed that the NaX-FAU particles cause a reduction of cell viability in a more pronounced way compared with the BEA particles. FROM THE CLINICAL EDITOR This article describes zeolite-based nanoparticles in generating time-controlled release of 5-FU from zeolite preparations for anti-cancer therapy.

Assembly of amelogenin proteolytic products and control of octacalcium phosphate crystal morphology.

The formation of enamel apatite crystals involves extracellular molecular events among which matrix assembly, interactions with growing crystals, and protein processing and removal are the subject of numerous investigations. Following the description of amelogenin nanospheres and the evidence for their presence in vivo as the principal structural component of developing dental enamel, we have focused our studies on investigating at the molecular level the process of nanosphere assembly and evaluating the effects of amelogenin on crystal growth and morphology. This paper is a short review of our recent studies with a focus on the assembly of amelogenin proteolytic products and their modulating effect on octacalcium phosphate (OCP) crystal morphology. In addition, we report that incorporation of amelogenins into 10% gelatin gel does not affect diffusion of calcium. This remarkable finding indicates that the observed modulation effect by amelogenin on OCP crystal morphology is not due to alteration of calcium diffusion into the gels but is the result of direct amelogenin-mineral interactions.

Merging high doxorubicin loading with pronounced magnetic response and bio-repellent properties in hybrid drug nanocarriers.

Hybrid magnetic drug nanocarriers are prepared via a self-assembly process of poly(methacrylic acid)-graft-poly(ethyleneglycol methacrylate) (p(MAA-g-EGMA)) on growing iron oxide nanocrystallites. The nanocarriers successfully merge together bio-repellent properties, pronounced magnetic response, and high loading capacity for the potent anticancer drug doxorubicin (adriamicin), in a manner not observed before in such hybrid colloids. High magnetic responses are accomplished by engineering the size of the magnetic nanocrystallites (∼13.5 nm) following an aqueous single-ferrous precursor route, and through adjustment of the number of cores in each colloidal assembly. Complementing conventional magnetometry, the magnetic response of the nanocarriers is evaluated by magnetophoretic experiments providing insight into their internal organization and on their response to magnetic manipulation. The structural organization of the graft-copolymer, locked on the surface of the nanocrystallites, is further probed by small-angle neutron scattering on single-core colloids. Analysis showed that the MAA segments selectively populate the area around the magnetic nanocrystallites, while the poly(ethylene glycol)-grafted chains are arranged as protrusions, pointing towards the aqueous environment. These nanocarriers are screened at various pHs and in highly salted media by light scattering and electrokinetic measurements. According to the results, their stability is dramatically enhanced, as compared to uncoated nanocrystallites, owing to the presence of the external protective PEG canopy. The nanocarriers are also endowed with bio-repellent properties, as evidenced by stability assays using human blood plasma as the medium.

Physicochemical characterization of solid dispersions of three antiepileptic drugs prepared by solvent evaporation method

We have investigated the solid dispersion and dissolution profiles of three antiepileptic drugs (carbamazepine (CBZ), oxcarbazepine (OXC) and rufinamide (RFN)) with different aqueous solubilities, prepared by the solvent evaporation method. Solid dispersions of the three drugs in hydroxy‐propylmethylcellulose (HPMC), with drug:polymer ratios of 1:4, were prepared and characterized by differential scanning calorimetry (DSC), Fourier transformation infrared (FTIR) spectroscopy, X‐ray diffraction (XRD) and scanning electron microscopy. The release mechanism was also investigated and the kinetic order of the solid dispersions was evaluated. It appeared that the dissolution behaviour depended on the physicochemical properties of the drug and drug‐polymer interactions. DSC thermographs showed amorphous forms for all drugs confirmed by XRD patterns. The FTIR spectra of CBZ and OXC demonstrated drug interactions with HPMC through hydrogen polymer bonds. Thus, solid dispersions of these drugs had an improved dissolution profile. In contrast, solid dispersions of RUF showed modest enhancement of dissolution, suggesting negligible drug‐polymer interactions. The different dissolution behaviour is attributed to the extent of interactions between the polymer hydroxyl group and the drug amide groups.

Colloidal stability of carbon nanotubes in an aqueous dispersion of phospholipid.

Within the family of nanomaterials, carbon nanotubes (CNTs) have emerged as a new efficient scaffold for studying molecular interactions at interfaces. Poor dispersability of CNTs in any solvent presents a considerable drawback for the development of novel functional composite structures. Previous studies have demonstrated that the solubility of CNTs can be greatly enhanced by employing appropriate surfactants, some of them being biological molecules. In this work, we study the noncovalent wrapping of lipid chains onto the graphitic surface of single-walled material (SWCNTs) by electron microscopy and Raman spectroscopy. Stable and homogenous aqueous suspensions of SWCNTs in the presence of lipids have been prepared, whereas their electrophoretic mobility was confirmed by ζ-potential measurements. Raman measurements revealed that smaller diameter SWCNTs are preferentially dispersed by lipid molecules in the aqueous supernatant part of the prepared suspension.We prepared stable homogeneous suspensions with layered double hydroxide (LDH) nanoparticles for in vitro gene delivery tests. The viability of HEK 293T cells in the presence of LDH nanoparticles at different concentrations was investigated. This revealed 50% cell viability at 500 μg/mL of LDH nanoparticles that is much higher than 50–100 μg/mL used for the delivery tests. The supercoiled pEF-eGFP plasmid (ca. 6100 base pairs) was mixed with LDH nanoparticle suspensions for anion exchange at a weight ratio of DNA/LDH between 1:25 and 1:100. In vitro experiments show that GFP expression in HEK 293T cells starts in the first day, reaches the maximum levels by the second day and continues in the third day. The GFP expression generally increases with the increase in DNA loading in DNA-LDH nanohybrids. However, the delivery efficiency with LDH nanoparticles as the agent is low. For example, the relative efficiency is 7%–15% of that of the commercial agent FuGENE®6. Three to 6% of total cells expressed GFP in an amount detectable by the FACS cytometry 2 days after transfection at 1 μg/mL of plasmid DNA with 25 μg/mL of LDH nanomaterial. The lower delivery efficiency could be attributed to the aggregation of LDH nanoparticles caused by the long-chain plasmid DNA.To enhance diagnostic or therapeutic efficacy, novel nanomaterials must be engineered to function in biologically relevant environments, be visible by conventional fluorescent microscopy, and have multivalent loading capacity for easy detection or effective drug delivery. Here we report the fabrication of silica nanoparticles doped with quantum dots and superficially functionalized with amino and phosphonate groups. The amino groups were acylated with a water-soluble biotin-labeling reagent. The biotinylated nanoparticles were subsequently decorated with neutravidin by exploiting the strong affinity between neutravidin and biotin. The resultant neutravidin-decorated fluorescent silica nanoparticles stably dispersed under physiological conditions, were visible by conventional optical and confocal fluorescent microscopy, and could be further functionalized with macromolecules, nucleic acids, and polymers. We also coated the surface of the nanoparticles with biotinylated mouse anti-human CD3 (αCD3). The resultant fluorescent nanoassembly was taken up by Jurkat T cells through receptor-mediated endocytosis and was partially released to lysosomes. Thus, quantum dot-doped silica nanoparticles decorated with neutravidin represent a potentially excellent scaffold for constructing specific intracellular nanoprobes and transporters.

Chitosan derivatives alter release profiles of model compounds from calcium phosphate implants.

The aim of the current study was to evaluate the impact of chitosan derivatives, namely N-octyl-chitosan and N-octyl-O-sulfate chitosan, incorporated in calcium phosphate implants to the release profiles of model drugs. The rate and extent of calcein (on M.W. 650 Da) ED, and FITC-dextran (M.W. 40 kDa) on in vitro release were monitored by fluorescence spectroscopy. Results show that calcein release is affected by the type of chitosan derivative used. A higher percentage of model drug was released when the hydrophilic polymer N-octyl-sulfated chitosan was present in the tablets compared with the tablets containing the hydrophobic polymer N-octyl-chitosan. The release profiles of calcein or FD from tablets containing N-octyl-O-sulfate revealed a complete release for FD after 120 h compared with calcein where 20% of the drug was released over the same time period. These results suggest that the difference in the release profiles observed from the implants is dependent on the molecular weight of the model drugs. These data indicate the potential of chitosan derivatives in controlling the release profile of active compounds from calcium phosphate implants.