The fate of nanocarriers as nanomedicines in vivo: important considerations and biological barriers to overcome.

Many pharmaceuticals on the market suffer from two significant limitations to their activity: lack of specificity toward the pathological site and poor aqueous solubility. Both factors therefore require the application of a large total dose of a drug to achieve high local concentration, causing numerous off-target toxic effects. Consequently, the grand aim of targeted drug delivery - the often-referred "magic bullet" - promises to improve drug concentration at the target site and maximize therapeutic response. Nanomaterial drug delivery systems have been explored extensively in the recent years for just this purpose. In the field of medicine, nanocarriers (NCs) have the potential to improve the biodistribution and pharmacokinetic characteristics of drugs, thereby reducing side effects while improving the therapeutic effect of drugs. Many nanomaterials are exquisitely designed and possess potent properties, yet it is extremely important to note that a general understanding of the interaction of nanomaterials with biological systems is essential for any such model properties to be effective in vivo, since the body presents a host of biological 'barriers' that will be encountered drug NCs. This review offers a general overview of the different biological obstacles that a NC must negotiate before it can carry out its desired role as a medicinal agent. From this standpoint we suggest aspects that should be considered for the rational design of novel nanomaterials possessing physicochemical properties that are appropriate for therapeutic or theragnostic applications.

Application of magnetically induced hyperthermia in the model protozoan Crithidia fasciculata as a potential therapy against parasitic infections.

BACKGROUND: Magnetic hyperthermia is currently a clinical therapy approved in the European Union for treatment of tumor cells, and uses magnetic nanoparticles (MNPs) under time-varying magnetic fields (TVMFs). The same basic principle seems promising against trypanosomatids causing Chagas disease and sleeping sickness, given that the therapeutic drugs available have severe side effects and that there are drug-resistant strains. However, no applications of this strategy against protozoan-induced diseases have been reported so far. In the present study, Crithidia fasciculata, a widely used model for therapeutic strategies against pathogenic trypanosomatids, was targeted with Fe(3)O(4) MNPs in order to provoke cell death remotely using TVMFs. METHODS: Iron oxide MNPs with average diameters of approximately 30 nm were synthesized by precipitation of FeSO(4) in basic medium. The MNPs were added to C. fasciculata choanomastigotes in the exponential phase and incubated overnight, removing excess MNPs using a DEAE-cellulose resin column. The amount of MNPs uploaded per cell was determined by magnetic measurement. The cells bearing MNPs were submitted to TVMFs using a homemade AC field applicator (f = 249 kHz, H = 13 kA/m), and the temperature variation during the experiments was measured. Scanning electron microscopy was used to assess morphological changes after the TVMF experiments. Cell viability was analyzed using an MTT colorimetric assay and flow cytometry. RESULTS: MNPs were incorporated into the cells, with no noticeable cytotoxicity. When a TVMF was applied to cells bearing MNPs, massive cell death was induced via a nonapoptotic mechanism. No effects were observed by applying TVMF to control cells not loaded with MNPs. No macroscopic rise in temperature was observed in the extracellular medium during the experiments. CONCLUSION: As a proof of principle, these data indicate that intracellular hyperthermia is a suitable technology to induce death of protozoan parasites bearing MNPs. These findings expand the possibilities for new therapeutic strategies combating parasitic infection.

Functionalized Fe3O4@Au superparamagnetic nanoparticles: in vitro bioactivity.

The interaction of nanoparticles with cells has been a focus of interest during the past decade. We report the fabrication and characterization of hydrosoluble Fe3O4@Au nanoparticles functionalized with biocompatible and fluorescent molecules and their interaction with cell cultures by visualizing them with confocal microscopy. Gold covered iron oxide nanoparticles were synthesized by reducing metal salts in the presence of oleylamine and oleic acid. The functionalization of these particles with an amphiphilic polymer provides a water soluble corona as well as the possibility to incorporate different molecules relevant for bio-applications such as poly(ethylene glycol), glucose or a cadaverine derived dye. The particle size, and the presence of polymer layers and conjugated molecules were characterized and confirmed by transmission electron microscopy, thermogravimetric measurements and infrared spectroscopy. A complete magnetic study was performed, showing that gold provides an optimum coating, which enhances the superparamagnetic behaviour observed above 10-15 K in this kind of nanoparticle. The interaction with cells and the cytotoxicity of the Fe3O4@Au preparations were determined upon incubation with the HeLa cell line. These nanoparticles showed no cytotoxicity when evaluated by the MTT assay and it was demonstrated that nanoparticles clearly interacted with the cells, showing a higher level of accumulation in the cells for glucose conjugated nanoparticles.

Advances in the design of new epoxy supports for enzyme immobilization-stabilization.

Multipoint covalent immobilization of enzymes (through very short spacer arms) on support surfaces promotes a very interesting 'rigidification' of protein molecules. In this case, the relative positions of each residue of the enzyme involved in the immobilization process have to be preserved unchanged during any conformational change induced on the immobilized enzyme by any distorting agent (heat, organic solvents etc.). In this way, multipoint covalent immobilization should induce a very strong stabilization of immobilized enzymes. Epoxy-activated supports are able to chemically react with all nucleophile groups placed on the protein surface: lysine, histidine, cysteine, tyrosine etc. Besides, epoxy groups are very stable. This allows the performance of very long enzyme-support reactions, enabling us to get very intense multipoint covalent attachment. In this way, these epoxy supports seem to be very suitable to stabilize industrial enzymes by multipoint covalent attachment. However, epoxy groups exhibit a low intermolecular reactivity towards nucleophiles and hence the enzymes are not able to directly react with the epoxy supports. Thus a rapid physical adsorption of enzymes on the supports becomes a first step, followed by an additional rapid 'intramolecular' reaction between the already adsorbed enzyme and the activated support. In this situation, a suitable first orientation of the enzyme on the support (e.g. through regions that are very rich in nucleophiles) is obviously necessary to get a very intense additional multipoint covalent immobilization. The preparation of different 'generations' of epoxy supports and the design of different protocols to fully control the first interaction between enzymes and epoxy supports will be reviewed in this paper. Finally, the possibilities of a directed immobilization of mutated enzymes (change of an amino acid by cysteine on specific points of the protein surface) on tailor-made disulfide-epoxy supports will be discussed as an almost-ideal procedure to achieve very intense and very efficient rigidification of a desired region of industrial enzymes.

Introduction of thiol-reactive structures on to soluble and insoluble proteins.

When proteins containing disulphide groups were oxidized with magnesium monoperoxyphthalate at acidic pH, they acquired the property of binding thiol compounds. This was the case with the insoluble protein keratin, chosen for having a large number of disulphide bridges, and with soluble ones like BSA and immunoglobulins. The potential applications of some of these modified proteins for the preparation of soluble bioconjugates have been explored. As a particular example of an application, the immobilization of activated IgG on to solid phases might provide a new way for preparing immunoadsorbents.