Potential role of calcifying nanoparticles in the etiology of multiple sclerosis.

Nanobacteria or calcifying nanoparticles are 80-500 nm sized nano-organisms that are physically associated with carbonate apatite mineral formations. They have been indicated in various diseases, including kidney stone formation, Alzheimer's disease, and atherosclerosis. Nanoparticles contain calcium and apatite-binding protein fetuin-A, a calcification inhibitor. However, recent evidence indicates that fetuin-A can form nucleation seeds or nidi that grow in size through ion sedimentation to become larger amorphous nanoparticles in the presence of excess calcium and apatite ions. Fetuin-A also functions as an inhibitor of meprin, a metalloproteinase implicated in inflammation and neurodegenerative diseases. During inflammation, meprin functions to regulate chemokine activity of monocyte chemotactic protein 1, which is associated with chronic inflammatory diseases, including atherosclerosis, renal inflammatory diseases, and multiple sclerosis (MS). In addition, calcium phosphate nanocrystals that contain fetuin-A are pro-inflammatory to macrophages and promote vascular smooth muscle cell mineralization, potentiating a vicious cycle of inflammation and calcification. Thus, mineral stress and inflammation appear to be associated with each other. Furthermore, fetuin-A deficient mice exhibited reduced experimental autoimmune encephalomyelitis severity. Thus, fetuin-A plays a direct role in the neuroinflammatory response. Indeed, the level of fetuin-A in cerebrospinal fluid has been defined as a biomarker of disease activity in MS. MS is a chronic, inflammatory, demyelinating, and neurodegenerative disease of the central nervous system (CNS) with an unknown etiology. The "inside-out" model of MS, supported by recent data, states that the initial axonal degeneration in the CNS occurs before demyelination, which then stimulates an auto-immune attack. It was shown very recently that influx of calcium from the extracellular space through nanoscale ruptures of the axonal plasma membrane predict axon degeneration in neuroinflammation. Calcium is an activator of calpains, proteases that function to break down the cytoskeleton, leading to neurodegeneration. Nanoruptures of the plasma membrane were suggested to occur at the early stages of axon damage, especially at nodes of Ranvier, which are devoid of myelin. Here, I propose that calcifying nanoparticles may have a role in the etiology and/or pathophysiology of MS. The initial event causing neurodegeneration may be due to the nanoparticles that have been suggested to easily cross the blood-brain barrier. Following this, the nanoparticles may create nanoruptures in the axonal membrane and also increase the calcium concentration around and within the neurons by forming nidi for calcification, eventually causing neurodegeneration. Nanoparticles can self-replicate; hence, they may represent an infectious causative agent for the development of MS.

Selenium nanoparticle as a bright promising anti-nanobacterial agent.

The use of nanotechnology for nanobacteria (or calcifying nanoparticles) treatment is a new creative approach. Use of selenium nanoparticles (SeNPs) as anti-nanobacterial agents might be considered as a bright promising approach due to their critical role in the inhibition of crystal growth and aggregation of calcium oxalate. Hence, in this study, we investigated the probable outcome of SeNPs inhibitory effects on growth of nanobacteria. Fragments of thirty urinary tract stones were chemically analyzed by X-ray diffraction (XRD) and urinary stones Kits for calcifying nanoparticles presence. Then powder of stone fragments were resuspended in Dulbecco's modified Eagle's medium (DMEM), sterilized by filtration and cultured in presence of 1, 5, 30, 60, and 90 µmol/L SeNPs concentrations. Besides, calcifying nanoparticles growth in the culture without SeNPs was measured spectrophotometrically. Also, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses were used, where calcifying nanoparticles formation occurred. Results showed that in the culture without SeNPs, the positive calcifying nanoparticles detection was 60% while after adding SeNPs at 90 µmol/L, not any calcifying nanoparticles were observed. Further confirmation came out when Energy-dispersive X-ray (EDX) analysis showed calcium and phosphate peaks in the culture medium without any SeNPs while in the culture containing 90 µm/L SeNPs a decrease in calcium and other minerals was obvious. Therefore, SeNPs clearly restricted the growth of nanobacteria due to their inhibitory effects on calcium oxalate deposition.

Visible light photocatalytic degradation of MB using UiO-66/g-C3N4 heterojunction nanocatalyst.

A unique hybrid of Zr-based metal-organic framework (UiO-66) with graphitic carbon nitride (g-C3N4) nanosheets was synthesized by a facile annealing method. Photocatalytic effect was measured by the photodegradation of methylene blue (MB) under visible light irradiation. The morphology, structure, and porous properties of the as-synthesized composites were characterized by using transmission electron microscopy (TEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), the thermogravimetric and differential scanning calorimetry analysis (TG-DSC), diffuse reflectance UV-vis spectroscopy (UV-vis DRS), photoluminescence (PL), and N2 sorption-desorption isotherms (BET). The results showed that about 100% of MB (200 mL of 10 mg L-1) photodegradation was achieved by the UiO-66/g-C3N4 hybrids (UC 10:10) in 240 min under visible light. The enhanced photocatalytic activity could be attributed to the heterojunction between UiO-66 and g-C3N4 therefore the photoelectron transfers efficiently from the conduction band (CB) of g-C3N4 to the CB of UiO-66 through the inner electric field generated by the heterojunction resulting the decreasing of recombination of electron/hole and the porous structures which enhance adsorption of the dye molecules on the catalyst surface thereby facilitates the electron/hole transfer within the framework. The trapping experiment and electron spin resonance (ESR) results showed that superoxide radicals (•O2-) was the main oxidative species in the photodegradation of MB and the enhanced photocatalytic mechanism of UiO-66/g-C3N4 heterojunction hybrids was also proposed.

Induction of apoptosis and autophagy by calcifying nanoparticles in human bladder cancer cells.

Calcifying nanoparticles have been linked to various types of human disease, but how they contribute to disease processes is unclear. Here, we examined whether and how calcifying nanoparticles isolated from patients with kidney stones are cytotoxic to human bladder cancer cells. Calcifying nanoparticles were isolated from midstream urine of patients with renal calcium oxalate stones and examined by electron microscopy. Human bladder cancer cells (EJ cells) were cultured in the presence of calcifying nanoparticles or nanohydroxyapatites for 12 and 72 h and examined for toxicity using the Cell Counting Kit-8, for autophagy using transmission electron microscopy and confocal microscopy, and for apoptosis using fluorescence microscopy, transmission electron microscopy, and flow cytometry. Changes in protein expression were analyzed by Western blotting. The results showed that the size and shape of the isolated calcifying nanoparticles were as expected. Calcifying nanoparticles were cytotoxic to EJ cells, more so than nanohydroxyapatites, and this was due, at least in part, to the production of intracellular reactive oxygen species. Transmission electron microscopy showed that calcifying nanoparticles were packaged into vesicles and autolysosomes. Calcifying nanoparticles induced greater autophagy and apoptosis than nanohydroxyapatites. Our findings demonstrate that calcifying nanoparticles can trigger bladder cancer cell injury by boosting reactive oxygen species production and stimulating autophagy and apoptosis.

Nanoparticle Tracking Analysis for the Enumeration and Characterization of Mineralo-Organic Nanoparticles in Feline Urine.

Urinary stone disease, particularly calcium oxalate, is common in both humans and cats. Calcifying nanoparticles (CNP) are spherical nanocrystallite material, and are composed of proteins (fetuin, albumin) and inorganic minerals. CNP are suggested to play a role in a wide array of pathologic mineralization syndromes including urolithiasis. We documented the development of a clinically relevant protocol to assess urinary CNP in 9 healthy cats consuming the same diet in a controlled environment using Nanoparticle Tracking Analysis (NTA®). NTA® is a novel method that allows for characterization of the CNP in an efficient, accurate method that can differentiate these particles from other urinary submicron particulates. The predominant nanoscale particles in feline urine are characteristic of CNP in terms of their size, their ability to spontaneously form under suitable conditions, and the presence of an outer layer that is rich in calcium and capable of binding to hydroxyapatite binders such as alendronate and osteopontin. The expansion of this particle population can be suppressed by the addition of citrate to urine samples. Further, compounds targeting exosomal surfaces do not label these particulates. As CNP have been associated with a number of significant urologic maladies, the method described herein may prove to be a useful adjunct in evaluating lithogenesis risk in mammals.

Of nanobacteria, nanoparticles, biofilms and their role in health and disease: facts, fancy and future.

Nanobacteria have been at the center of a major scientific controversy in recent years owing to claims that they represent not only the smallest living microorganisms on earth but also new emerging pathogens associated with several human diseases. We and others have carefully examined these claims and concluded that nanobacteria are in fact nonliving mineralo-organic nanoparticles (NPs) that form spontaneously in body fluids. We have shown that these mineral particles possess intriguing biomimetic properties that include the formation of cell- and tissue-like morphologies and the possibility to grow, proliferate and propagate by subculture. Similar mineral NPs (bions) have now been found in both physiological and pathological calcification processes and they appear to represent precursors of physiological calcification cycles, which may at times go awry in disease conditions. Furthermore, by functioning at the nanoscale, these mineralo-organic NPs or bions may shed light on the fate of nanomaterials in the body, from both nanotoxicological and nanopathological perspectives.

Clinical implications of calcifying nanoparticles in dental diseases: a critical review.

BACKGROUND: Unknown cell-culture contaminants were described by Kajander and Ciftçioglu in 1998. These contaminants were called nanobacteria initially and later calcifying nanoparticles (CNPs). Their exact nature is unclear and controversial. CNPs have unique and unusual characteristics, which preclude placing them into any established evolutionary branch of life. AIM: The aim of this systematic review was to assess published data concerning CNPs since 1998 in general and in relation to dental diseases in particular. MATERIALS AND METHODS: The National Library of Medicine (PubMed) and Society of Photographic Instrumentation Engineers (SPIE) electronic and manual searches were conducted. Nanobacteria and calcifying nanoparticles were used as keywords. The search yielded 135 full-length papers. Further screening of the titles and abstracts that followed the review criteria resulted in 43 papers that met the study aim. CONCLUSION: The review showed that the existence of nanobacteria is still controversial. Some investigators have described a possible involvement of CNPs in pulpal and salivary gland calcifications, as well as the possible therapeutic use of CNPs in the treatment of cracked and/or eroded teeth.

Bioactive macro/micro porous silk fibroin/nano-sized calcium phosphate scaffolds with potential for bone-tissue-engineering applications.

AIM: The development of novel silk/nano-sized calcium phosphate (silk/nano-CaP) scaffolds with highly dispersed CaP nanoparticles in the silk fibroin (SF) matrix for bone tissue engineering. MATERIALS & METHODS: Nano-CaP was incorporated in a concentrated aqueous SF solution (16 wt.%) by using an in situ synthesis method. The silk/nano-CaP scaffolds were then prepared through a combination of salt-leaching/lyophilization approaches. RESULTS: The CaP particles presented good affinity to SF and their size was inferior to 200 nm when theoretical CaP/silk ratios were between 4 and 16 wt.%, as determined by scanning electron microscopy. The CaP particles displayed a uniform distribution in the scaffolds at both microscopic and macroscopic scales as observed by backscattered scanning electron microscopy and micro-computed tomography, respectively. The prepared scaffolds presented self-mineralization capability and no cytotoxicity confirmed by in vitro bioactivity tests and cell viability assays, respectively. CONCLUSION: These results indicated that the produced silk/nano-CaP scaffolds could be suitable candidates for bone-tissue-engineering applications.

A silicon cell cycle in a bacterial model of calcium phosphate mineralogenesis.

The prokaryote Corynebacterium matruchotii produces calcium phosphate (bone salt) and may serve as a convenient model for examining individual factors relevant to vertebrate calcification. A factor of current clinical uncertainty is silicon. To investigate its possible role in biomineralisation advanced optical (digital deconvolution and 3D fluorescent image rendering) and electron microscopy (EDX microanalysis and elemental mapping) were applied to calcifying microbial colonies grown in graded Si concentrations (0-60mM). Cell viability was confirmed throughout by TO-PRO-3-iodide and SYTO-9 nucleic acid staining. It was observed that calcium accumulated in dense intracellular microspherical objects (types i-iii) as nanoparticles (5 nm, type i), nanospheres (30-50 nm, type ii) and filamentous clusters (0.1-0.5 µm, type iii), with a regular transitory Si content evident. With bacterial colony development (7-28 days) the P content increased from 5 to 60%, while Si was displaced from 60 to 5%, distinguishing the phenomenon from random contamination, and with a significant relationship (p<0.001) found between calcified object number and Si supplementation (optimum 0.01mM). The Si-containing, intracellular calcified objects (also positive for Mg and negative with Lysensor blue DND-167 for acidocalcisomes) were extruded naturally in bubble-like chains to complete the cycle by coating the cell surface with discrete mineral particles. These could be harvested by lysis, French press and density fractionation when Si was confirmed in a proportion. It was concluded that the unexplained orthopaedic activity of Si may derive from its special property to facilitate calcium phosphorylation in biological systems, thereby recapitulating an ancient and conserved bacterial cycle of calcification via silicification.

Order-disorder transition of aragonite nanoparticles in nacre.

Understanding nacre's bottom-up biomineralization mechanism, particularly, how individual aragonite platelets are formed, has long remained elusive due to its crystallographic peculiarity and structural complexity. Here we report that crystallographic order-disorder transition can be triggered within individual aragonite platelets in pristine nacre by means of heat treatment and/or inelastic deformation, offering a unique opportunity to discriminate mysterious aragonite nanoparticles in transmission electron microscopy. Our findings unambiguously uncover why aragonite nanoparticles in pristine nacre have long been inaccessible under TEM observation, which is attributed to the monocrystal-polycrystal duality of the aragonite platelet. The underlying physical mechanism for why an individual aragonite platelet adopts a highly oriented attachment of aragonite nanoparticles as its crystallization pathway is, for the first time, explained in terms of the thermodynamics. The finding of an order-disorder transition in nacre provides a new perspective for understanding the formation for other biominerals.

Hemoglobin aggregates studied under static and dynamic conditions involving the formation of nanobacteria-like structures.

Laser light scattering and scanning electron microscopy (SEM) are used to study hemoglobin in the aqueous phase. The impact that salts [NaCl, Ca3(PO4)2] and iron oxide nanoparticles have on the hemoglobin size are also studied. The first set of experiments examined hemoglobin aggregates in the aqueous phases in the presence of salts and nanoparticles. Aqueous phase samples were then dehydrated and examined using SEM. The resulting structures resemble those observed in nanobacteria studies conducted in other labs. This study demonstrates that aggregates of hemoglobin and various salts found in a physiological environment can produce structures that resemble nanobacteria.

The role of calcifying nanoparticles in biology and medicine.

Calcifying nanoparticles (CNPs) (nanobacteria, nanobacteria-like particles, nanobes) were discovered over 25 years ago; nevertheless, their nature is still obscure. To date, nobody has been successful in credibly determining whether they are the smallest self-replicating life form on Earth, or whether they represent mineralo-protein complexes without any relation to living organisms. Proponents of both theories have a number of arguments in favor of the validity of their hypotheses. However, after epistemological analysis carried out in this review, all arguments used by proponents of the theory about the physicochemical model of CNP formation may be refuted on the basis of the performed investigations, and therefore published data suggest a biological nature of CNPs. The only obstacle to establish CNPs as living organisms is the absence of a fairly accurately sequenced genome at the present time. Moreover, it is clear that CNPs play an important role in etiopathogenesis of many diseases, and this association is independent from their nature. Consequently, emergence of CNPs in an organism is a pathological, not a physiological, process. The classification and new directions of further investigations devoted to the role of CNPs in biology and medicine are proposed.