Activity of Antibiotics and Potential Antibiofilm Agents against Biofilm-Producing Mycobacterium avium-intracellulare Complex Causing Chronic Pulmonary Infections.

Nontuberculous mycobacteria (NTM) cause lung infections in patients with underlying pulmonary diseases (PD). The Mycobacteriumavium-intracellulare complex (MAC) is the most frequently involved NTM. The MAC-PD treatment is based on the administration of several antibiotics for long periods of time. Nonetheless, treatment outcomes remain very poor. Among the factors involved is the ability of MAC isolates to form biofilm. The aim of the study was to assess the in vitro activity of different antibiotics and potential antibiofilm agents (PAAs) against MAC biofilm. Four antibiotics and six PAAs, alone and/or in combination, were tested against planktonic forms of 11 MAC clinical isolates. Biofilm was produced after 4 weeks of incubation and analyzed with the crystal violet assay. The antibiotics and PAAs were tested by measuring the absorbance (minimum biofilm inhibition concentrations, MBICs) and by performing subcultures (minimum biofilm eradication concentrations, MBECs). The clarithromycin/amikacin and clarithromycin/ethambutol combinations were synergistic, decreasing the MBECs values compared to the individual antibiotics. The amikacin/moxifloxacin combination showed indifference. The MBIC values decreased significantly when PAAs were added to the antibiotic combinations. These results suggest that antibiotic combinations should be further studied to establish their antibiofilm activity. Moreover, PAAs could act against the biofilm matrix, facilitating the activity of antibiotics.

Prussian Blue: A Nanozyme with Versatile Catalytic Properties.

Nanozymes, nanomaterials with enzyme-like activities, are becoming powerful competitors and potential substitutes for natural enzymes because of their excellent performance. Nanozymes offer better structural stability over their respective natural enzymes. In consequence, nanozymes exhibit promising applications in different fields such as the biomedical sector (in vivo diagnostics/and therapeutics) and the environmental sector (detection and remediation of inorganic and organic pollutants). Prussian blue nanoparticles and their analogues are metal-organic frameworks (MOF) composed of alternating ferric and ferrous irons coordinated with cyanides. Such nanoparticles benefit from excellent biocompatibility and biosafety. Besides other important properties, such as a highly porous structure, Prussian blue nanoparticles show catalytic activities due to the iron atom that acts as metal sites for the catalysis. The different states of oxidation are responsible for the multicatalytic activities of such nanoparticles, namely peroxidase-like, catalase-like, and superoxide dismutase-like activities. Depending on the catalytic performance, these nanoparticles can generate or scavenge reactive oxygen species (ROS).

Dual Effect of Prussian Blue Nanoparticles on Aß40 Aggregation: ß-Sheet Fibril Reduction and Copper Dyshomeostasis Regulation.

Alzheimer's disease (AD), affecting almost 50 million individuals worldwide, is currently the first cause of dementia. Despite the tremendous research efforts in the last decade, only four supportive or palliative drugs, namely, acetylcholinesterase (AChE) inhibitors donepezil, galantamine, and rivastigmine and the glutamate NMDA receptor antagonist memantine, are currently available. New therapeutic strategies are becoming prominent, such as the direct inhibition of amyloid formation or the regulation of metal homeostasis. In the present report, the potential use of Prussian blue (PB), a drug that is in the World Health Organization Model List of Essential Medicines, in AD treatment is demonstrated. Both in vitro and in cellulo studies indeed suggest that PB nanoparticles (PBNPs) are capable of reducing the formation of typical amyloid-ß fibers (detected by thioflavin T fluorescence) and restoring the usual amyloid fibrillation pathway via chelation/sequestration of copper, which is found in high concentrations in senile plaques.

Prussian Blue: A Safe Pigment with Zeolitic-Like Activity.

Prussian blue (PB) and PB analogues (PBA) are coordination network materials that present important similarities with zeolites concretely with their ability of adsorbing cations. Depending on the conditions of preparation, which is cheap and easy, PB can be classified into soluble PB and insoluble PB. The zeolitic-like properties are mainly inherent to insoluble form. This form presents some defects in its cubic lattice resulting in an open structure. The vacancies make PB capable of taking up and trapping ions or molecules into the lattice. Important adsorption characteristics of PB are a high specific area (370 m2 g-1 determined according the BET theory), uniform pore diameter, and large pore width. PB has numerous applications in many scientific and technological fields. PB are assembled into nanoparticles that, due to their biosafety and biocompatibility, can be used for biomedical applications. PB and PBA have been shown to be excellent sorbents of radioactive cesium and radioactive and nonradioactive thallium. Other cations adsorbed by PB are K+, Na+, NH4+, and some divalent cations. PB can also capture gaseous molecules, hydrocarbons, and even luminescent molecules such as 2-aminoanthracene. As the main adsorptive application of PB is the selective removal of cations from the environment, it is important to easily separate the sorbent of the purified solution. To facilitate this, PB is encapsulated into a polymer or coats a support, sometimes magnetic particles. Finally, is remarkable to point out that PB can be recycled and the adsorbed material can be recovered.

Superparamagnetic Nanoparticles with Efficient Near-Infrared Photothermal Effect at the Second Biological Window.

Superparamagnetic nanoparticles (iron oxide nanoparticles-IONs) are suitable for hyperthermia after irradiating with radiofrequency radiation. Concerning the suitability for laser ablation, IONs present a low molar absorption coefficient in the near-infrared region close to 800 nm. For this reason, they are combined with other photothermal agents into a hybrid composite. Here, we show that IONs absorb and convert into heat the infrared radiation characteristic of the so-called second-biological window (1000-1350 nm) and, in consequence, they can be used for thermal ablation in such wavelengths. To the known excellent water solubility, colloidal stability and biocompatibility exhibited by IONs, an outstanding photothermal performance must be added. For instance, a temperature increase of 36 °C was obtained after irradiating at 8.7 W cm-2 for 10 min a suspension of IONs at iron concentration of 255 mg L-1. The photothermal conversion efficiency was ~72%. Furthermore, IONs showed high thermogenic stability during the whole process of heating/cooling. To sum up, while the use of IONs in the first bio-window (700-950 nm) presents some concerns, they appear to be good photothermal agents in the second biological window.

Flash tooth whitening: A friendly formulation based on a nanoencapsulated reductant.

Tooth whitening materials have not undergone relevant advances in the last years. Current materials base their action on the oxidant activity of peroxides, which present the disadvantage of requiring long application times, along with unpleasant side effects of dental hypersensitivity (e.g. sharp pain). In this work, a novel tooth whitening formulation based on the encapsulation of a reducing agent (sodium metabisulfite) in liposomes is developed. An experimental design was applied to optimize the formulation in terms of whitening action and safety, using bovine teeth as in vitro model. Results were obtained by colorimetry, profilometry and nanoindentation techniques. The comparison with standard whitening treatments showed a similar whitening action of the optimized formulation but in remarkable shorter application times. Moreover, teeth roughness values obtained with the presented formulation conformed with ISO 28399. As mechanism of action, results obtained from fluorescent confocal microscopy showed the liposomal formulation to form a layer surrounding the enamel surface, enhancing the treatment efficacy in terms of diffusion of the protected reductant towards the enamel. The better efficiency of this formulation encourages its use as an alternative to current oxidative treatments.

Prussian blue nanoparticles: synthesis, surface modification, and biomedical applications.

Prussian blue nanoparticles (PBNPs) are a nanomaterial that presents unique properties and an excellent biocompatibility. They can be synthesized in mild conditions and can be derivatized with polymers and/or biomolecules. PBNPs are used in biomedicine as therapy and diagnostic agents. In biomedical imaging, PBNPs constitute contrast agents in photoacoustic and magnetic resonance imaging (MRI). They are a good adsorbent to be used as antidotes for poisoning with cesium and/or thallium ions. Moreover, the ability to convert energy into heat makes them useful photothermal agents (PAs) in photothermal therapy (PTT) or as nonantibiotic substances with antibacterial properties. Finally, PBNPs can be both reduced to Prussian white and oxidized to Prussian green. A large window of redox potential exists between reduction and oxidation, which result in the enzyme-like characteristics of these NPs.

Facile Synthesis of Novel Prussian Blue-Lipid Nanocomplexes.

Prussian blue (PB) is known for its multiple applications ranging from fine arts to therapeutics. More recently, PB nanoparticles have been pointed to as appealing photothermal agents (PA) when irradiated with wavelengths corresponding to the biological windows, namely regions located in the near infrared (NIR) zone. In addition, the combination of PB with other components such as phospholipids boosts their therapeutical potential by facilitating, for instance, the incorporation of drugs becoming suitable drug delivery systems. The novelty of the research relies on the synthesis procedure and characterization of hybrid lipid-PB nanoparticles with a high yield in a friendly environment suitable for photothermal therapy. This goal was achieved by first obtaining insoluble PB coated with oleylamine (OA) to facilitate its combination with lipids. The resulting lipid-PB complex showed a monomodal distribution of sizes with an overall size of around 100 nm and a polydispersity index of about 0.200. It highlights one critical step in the synthesis procedure that is the shaking time of the mixture of PB-OA nanoparticles with the lipid, which was found to be 48 h. This time assured homogeneous preparation without the need of further separation stages. Samples were stable for more than three months under several storage conditions.

Bacterial Inclusion Bodies for Anti-Amyloid Drug Discovery: Current and Future Screening Methods.

Amyloid aggregation is linked to an increasing number of human disorders from nonneurological pathologies such as type-2 diabetes to neurodegenerative ones such as Alzheimer or Parkinson's diseases. Thirty-six human proteins have shown the capacity to aggregate into pathological amyloid structures. To date, it is widely accepted that amyloid folding/aggregation is a universal process present in eukaryotic and prokaryotic cells. In the last decade, several studies have unequivocally demonstrated that bacterial inclusion bodies - insoluble protein aggregates usually formed during heterologous protein overexpression in bacteria - are mainly composed of overexpressed proteins in amyloid conformation. This fact shows that amyloid-prone proteins display a similar aggregation propensity in humans and bacteria, opening the possibility to use bacteria as simple models to study amyloid aggregation process and the potential effect of both anti-amyloid drugs and pro-aggregative compounds. Under these considerations, several in vitro and in cellulo methods, which exploit the amyloid properties of bacterial inclusion bodies, have been proposed in the last few years. Since these new methods are fast, simple, inexpensive, highly reproducible, and tunable, they have aroused great interest as preliminary screening tools in the search for anti-amyloid (beta-blocker) drugs for conformational diseases. The aim of this mini-review is to compile recently developed methods aimed at tracking amyloid aggregation in bacteria, discussing their advantages and limitations, and the future potential applications of inclusion bodies in anti-amyloid drug discovery.

Iron Oxide Nanoparticles in Photothermal Therapy.

Photothermal therapy is a kind of therapy based on increasing the temperature of tumoral cells above 42 °C. To this aim, cells must be illuminated with a laser, and the energy of the radiation is transformed in heat. Usually, the employed radiation belongs to the near-infrared radiation range. At this range, the absorption and scattering of the radiation by the body is minimal. Thus, tissues are almost transparent. To improve the efficacy and selectivity of the energy-to-heat transduction, a light-absorbing material, the photothermal agent, must be introduced into the tumor. At present, a vast array of compounds are available as photothermal agents. Among the substances used as photothermal agents, gold-based compounds are one of the most employed. However, the undefined toxicity of this metal hinders their clinical investigations in the long run. Magnetic nanoparticles are a good alternative for use as a photothermal agent in the treatment of tumors. Such nanoparticles, especially those formed by iron oxides, can be used in combination with other substances or used themselves as photothermal agents. The combination of magnetic nanoparticles with other photothermal agents adds more capabilities to the therapeutic system: the nanoparticles can be directed magnetically to the site of interest (the tumor) and their distribution in tumors and other organs can be imaged. When used alone, magnetic nanoparticles present, in theory, an important limitation: their molar absorption coefficient in the near infrared region is low. The controlled clustering of the nanoparticles can solve this drawback. In such conditions, the absorption of the indicated radiation is higher and the conversion of energy in heat is more efficient than in individual nanoparticles. On the other hand, it can be designed as a therapeutic system, in which the heat generated by magnetic nanoparticles after irradiation with infrared light can release a drug attached to the nanoparticles in a controlled manner. This form of targeted drug delivery seems to be a promising tool of chemo-phototherapy. Finally, the heating efficiency of iron oxide nanoparticles can be increased if the infrared radiation is combined with an alternating magnetic field.

Reversible and irreversible aggregation of magnetic liposomes.

Understanding stabilization and aggregation in magnetic nanoparticle systems is crucial to optimizing the functionality of these systems in real physiological applications. Here we address this problem for a specific, yet representative, system. We present an experimental and analytical study on the aggregation of superparamagnetic liposomes in suspension in the presence of a controllable external magnetic field. We study the aggregation kinetics and report an intermediate time power law evolution and a long time stationary value for the average aggregate diffusion coefficient, both depending on the magnetic field intensity. We then show that the long time aggregate structure is fractal with a fractal dimension that decreases upon increasing the magnetic field intensity. By scaling arguments we also establish an analytical relation between the aggregate fractal dimension and the power law exponent controlling the aggregation kinetics. This relation is indeed independent on the magnetic field intensity. Despite the superparamagnetic character of our particles, we further prove the existence of a population of surviving aggregates able to maintain their integrity after switching off the external magnetic field. Finally, we suggest a schematic interaction scenario to rationalize the observed coexistence between reversible and irreversible aggregation.

Magnetic Nanoemulsions: Comparison between Nanoemulsions Formed by Ultrasonication and by Spontaneous Emulsification.

Nanoemulsions are particularly suitable as a platform in the development of delivery systems. The type of nanoemulsion with a higher stability will offer an advantage in the preparation of a delivery system for lipophilic drugs. Nanoemulsions can be fabricated by different processing methods, which are usually categorized as either high- or low-energy methods. In this study, a comparison between two methods of preparing magnetic oil-in-water (O/W) nanoemulsions is described. The nanoemulsions were formed by sonication (the high-energy method) or by spontaneous emulsification (the low-energy method). In both cases, the oil phase was olive oil, and a phospholipid and a pegylated phospholipid were used as emulsifiers. To favor the comparison, the amounts of the components were the same in both kinds of nanoemulsions. Moreover, nanoemulsions were loaded with hydrophobic superparamagnetic nanoparticles and indomethacin. In vitro, releases studies indicated a short drug burst period followed by a prolonged phase of dissolutive drug release. The Korsmeyer-Peppas model can fit the associated kinetics. The results showed that such nanoemulsions are suitable as a platform in the development of delivering systems for lipophilic drugs. The long-term stability was also examined at different temperatures, as well as the interaction with plasma proteins. Nanoemulsion obtained by the low-energy method showed a great stability at 4 °C and at ambient temperature. Its size and polydispersity did not change over more than two months. The spontaneous emulsification method therefore has great potential for forming nanoemulsion-based delivery systems.

Evidence of Protein Adsorption in Pegylated Liposomes: Influence of Liposomal Decoration.

In order to contribute to a better knowledge of the events involved in the formation of the protein corona when nanoparticles (NPs) come in contact with proteins, we report a study about the changes on the physicochemical properties of pristine, PEGylated and Cyclic Arginine-Glycine-Aspartate peptide (RGD)-functionalized large unilamelar liposomes (LUVs) or magnetoliposomes (MLs) upon incubation with Bovine Serum Albumin (BSA). The main phospholipid component of both LUVs and MLs was l-α-phosphatydylcholine (PC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) with 20% of cholesterol. The most obvious indication of the interaction of BSA-nanosystems is given by changes in the hydrodynamic diameter of the particles but other evidence is needed to corroborate the process. Our findings indicate that size modification is a process that is accomplished in few hours and that is strongly dependent not only on the surface decoration but also of the lipid composition of both LUVs and MLs. Fluorescence quenching experiments as well as cryogenic transmission electron microscopy (Cryo-TEM) images assessed these changes and confirmed that although each system has to be studied in a particular way, we can establish three distinctive features that turn into more reactive systems: (a) compositions containing PC compared with their DMPC counterparts; (b) the presence of PEG and/or RGD compared to the pristine counterparts; and (c) the presence of SPIONs: MLs show higher interaction than LUVs of the same lipid composition. Consequently, PEGylation (that is supposed to make stealth NPs) actually fails in preventing complete protein binding.

Combined in Vitro Cell-Based/in Silico Screening of Naturally Occurring Flavonoids and Phenolic Compounds as Potential Anti-Alzheimer Drugs.

Alzheimer's disease (AD) is the main cause of dementia in people over 65 years. One of the major culprits in AD is the self-aggregation of amyloid-ß peptide (Aß), which has stimulated the search for small molecules able to inhibit Aß aggregation. In this context, we recently reported a simple, but effective in vitro cell-based assay to evaluate the potential antiaggregation activity of putative Aß aggregation inhibitors. In this work this assay was used together with docking and molecular dynamics simulations to analyze the anti-Aß aggregation activity of several naturally occurring flavonoids and phenolic compounds. The results showed that rosmarinic acid, melatonin, and o-vanillin displayed zero or low inhibitory capacity, curcumin was found to have an intermediate inhibitory potency, and apigenin and quercetin showed potent antiaggregation activity. Finally, the suitability of the combined in vitro cell-based/in silico approach to distinguish between active and inactive compounds was further assessed for an additional set of flavonols and dihydroflavonols.

Effect of PEGylation on Ligand-Targeted Magnetoliposomes: A Missed Goal.

We tested the targeting efficiency of magnetoliposomes (MLPs) labeled with tripeptide arginine-glycine-aspartic acid (RGD) on two types of cells: HeLa cells expressing RGD receptors and 3T3 cells lacking RGD receptors. The targeting ability of RGD-MLPs was compared to that of bare MLPs and MLPs stabilized with poly(ethylene glycol) (PEG). Cellular internalization of these liposomes was determined by flow cytometry and confocal microscopy, which showed that both types of cells took up more nontargeting MLPs than targeting RGD-MLPs or PEG-MLPs, with PEG-MLPs showing the lowest degree of internalization. The presence of specific receptors on HeLa cells did not facilitate the binding of RGD-MLPs, probably due to the presence of PEG chains on the liposomal surface. The polymer increases the circulation time of the liposomes in the organism but reduces their interactions with cells. Despite the localization of the RGD peptide on the tip of PEG in RGD-MLPs, the interaction between the liposome and cell was still limited. To avoid this drawback, targeting drug delivery systems can be prepared with two types of PEG: one of a short length to enable biocompatibility and the other of a longer chain to carry the ligand.

Oil-in-water nanoemulsions are suitable for carrying hydrophobic compounds: Indomethacin as a model of anti-inflammatory drug.

Oil-in-water nanoemulsions are increasingly being used as delivery systems for encapsulating lipophilic components in functional food, personal care and pharmaceutical products. In the current study, we developed a multimodal platform to carry hydrophobic indomethacin or magnetic nanoparticles, or both. As a consequence, this platform has great potential for therapeutic or imaging purposes. By optimizing the system composition and homogenization conditions, a nanoemulsion with a mean droplet diameter of about 200nm and a low polydispersity index (<0.2) was formed. The plain nanoemulsion was shown to be innocuous in cellular studies and did not present acute toxicity (observed in a rat model). More interesting was the finding that nanoemulsions loaded with indomethacin presented a significantly different anti-inflammatory than the free drug.

Liposomes Loaded with Hydrophobic Iron Oxide Nanoparticles: Suitable T2 Contrast Agents for MRI.

There has been a recent surge of interest in the use of superparamagnetic iron oxide nanoparticles (SPIONs) as contrast agents (CAs) for magnetic resonance imaging (MRI), due to their tunable properties and their low toxicity compared with other CAs such as gadolinium. SPIONs exert a strong influence on spin-spin T2 relaxation times by decreasing the MR signal in the regions to which they are delivered, consequently yielding darker images or negative contrast. Given the potential of these nanoparticles to enhance detection of alterations in soft tissues, we studied the MRI response of hydrophobic or hydrophilic SPIONs loaded into liposomes (magnetoliposomes) of different lipid composition obtained by sonication. These hybrid nanostructures were characterized by measuring several parameters such as size and polydispersity, and number of SPIONs encapsulated or embedded into the lipid systems. We then studied the influence of acyl chain length as well as its unsaturation, charge, and presence of cholesterol in the lipid bilayer at high field strength (7 T) to mimic the conditions used in preclinical assays. Our results showed a high variability depending on the nature of the magnetic particles. Focusing on the hydrophobic SPIONs, the cholesterol-containing samples showed a slight reduction in r2, while unsaturation of the lipid acyl chain and inclusion of a negatively charged lipid into the bilayer appeared to yield a marked increase in negative contrast, thus rendering these magnetoliposomes suitable candidates as CAs, especially as a liver CA.

Key Points Concerning Amyloid Infectivity and Prion-Like Neuronal Invasion.

Amyloid aggregation has been related to an increasing number of human illnesses, from Alzheimer's and Parkinson's diseases (AD/PD) to Creutzfeldt-Jakob disease. Commonly, only prions have been considered as infectious agents with a high capacity of propagation. However, recent publications have shown that many amyloid proteins, including amyloid ß-peptide, α-synuclein (α-syn) and tau protein, also propagate in a "prion-like" manner. Meanwhile, no link between propagation of pathological proteins and neurotoxicity has been demonstrated. The extremely low infectivity under natural conditions of most non-prion amyloids is far below the capacity to spread exhibited by prions. Nonetheless, it is important to elucidate the key factors that cause non-prion amyloids to become infectious agents. In recent years, important advances in our understanding of the amyloid processes of amyloid-like proteins and unrelated prions (i.e., yeast and fungal prions) have yielded essential information that can shed light on the prion phenomenon in mammals and humans. As shown in this review, recent evidence suggests that there are key factors that could dramatically modulate the prion capacity of proteins in the amyloid conformation. The concentration of nuclei, the presence of oligomers, and the toxicity, resistance and localization of these aggregates could all be key factors affecting their spread. In short, those factors that favor the high concentration of extracellular nuclei or oligomers, characterized by small size, with a low toxicity could dramatically increase prion propensity; whereas low concentrations of highly toxic intracellular amyloids, with a large size, would effectively prevent infectivity.

Amyloids in solid-state nuclear magnetic resonance: potential causes of the usually low resolution.

Amyloids are non-crystalline and insoluble, which imply that the classical structural biology tools, ie, X-ray crystallography and solution nuclear magnetic resonance (NMR), are not suitable for their analysis. In the last years, solid-state NMR (ssNMR) has emerged as an alternative tool to decrypt the structural signatures of amyloid fibrils, providing major contributions to our understanding of molecular structures of amyloids such as ß-amyloid peptide associated with Alzheimer's disease or fungal prions, among others. Despite this, the wide majority of amyloid fibrils display low resolution by ssNMR. Usually, this low resolution has been attributed to a high disorder or polymorphism of the fibrils, suggesting the existence of diverse elementary ß-sheet structures. Here, we propose that a single ß-sheet structure could be responsible for the broadening of the line widths in the ssNMR spectra. Although the fibrils and fibers consist of a single elementary structure, the angle of twist of each individual fibril in the mature fiber depends on the number of individual fibrils as well as the fibril arrangement in the final mature fiber. Thus, a wide range of angles of twist could be observed in the same amyloid sample. These twist variations involve changes in amino acid alignments that could be enough to limit the ssNMR resolution.

Iron oxide nanoparticles for magnetically-guided and magnetically-responsive drug delivery.

In this review, we discuss the recent advances in and problems with the use of magnetically-guided and magnetically-responsive nanoparticles in drug delivery and magnetofection. In magnetically-guided nanoparticles, a constant external magnetic field is used to transport magnetic nanoparticles loaded with drugs to a specific site within the body or to increase the transfection capacity. Magnetofection is the delivery of nucleic acids under the influence of a magnetic field acting on nucleic acid vectors that are associated with magnetic nanoparticles. In magnetically-responsive nanoparticles, magnetic nanoparticles are encapsulated or embedded in a larger colloidal structure that carries a drug. In this last case, an alternating magnetic field can modify the structure of the colloid, thereby providing spatial and temporal control over drug release.