Quantitative Determination of Skin Penetration of PEG-Coated CdSe Quantum Dots in Dermabraded but not Intact SKH-1 Hairless Mouse Skin

Putative living entities called nanobacteria (NB) are unusual for their small sizes (50–500 nm), pleomorphic nature, and accumulation of hydroxyapatite (HAP), and have been implicated in numerous diseases involving extraskeletal calcification. By adding precipitating ions to cell culture medium containing serum, mineral nanoparticles are generated that are morphologically and chemically identical to the so-called NB. These nanoparticles are shown here to be formed of amorphous mineral complexes containing calcium as well as other ions like carbonate, which then rapidly acquire phosphate, forming HAP. The main constituent proteins of serum-derived NB are albumin, fetuin-A, and apolipoprotein A1, but their involvement appears circumstantial since so-called NB from different body fluids harbor other proteins. Accordingly, by passage through various culture media, the protein composition of these particles can be modulated. Immunoblotting experiments reveal that antibodies deemed specific for NB react in fact with either albumin, fetuin-A, or both, indicating that previous studies using these reagents may have detected these serum proteins from the same as well as different species, with human tissue nanoparticles presumably absorbing bovine serum antigens from the culture medium. Both fetal bovine serum and human serum, used earlier by other investigators as sources of NB, paradoxically inhibit the formation of these entities, and this inhibition is trypsin-sensitive, indicating a role for proteins in this inhibitory process. Fetuin-A, and to a lesser degree albumin, inhibit nanoparticle formation, an inhibition that is overcome with time, ending with formation of the so-called NB. Together, these data demonstrate that NB are most likely formed by calcium or apatite crystallization inhibitors that are somehow overwhelmed by excess calcium or calcium phosphate found in culture medium or in body fluids, thereby becoming seeds for calcification. The structures described earlier as NB may thus represent remnants and by-products of physiological mechanisms used for calcium homeostasis, a concept which explains the vast body of NB literature as well as explains the true origin of NB as lifeless protein-mineralo entities with questionable role in pathogenesis.

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Putative Nanobacteria Represent Physiological Remnants and Culture By-Products of Normal Calcium Homeostasis

Calcium and apatite granulations are demonstrated here to form in both human and fetal bovine serum in response to the simple addition of either calcium or phosphate, or a combination of both. These granulations are shown to represent precipitating complexes of protein and hydroxyapatite (HAP) that display marked pleomorphism, appearing as round, laminated particles, spindles, and films. These same complexes can be found in normal untreated serum, albeit at much lower amounts, and appear to result from the progressive binding of serum proteins with apatite until reaching saturation, upon which the mineralo-protein complexes precipitate. Chemically and morphologically, these complexes are virtually identical to the so-called nanobacteria (NB) implicated in numerous diseases and considered unusual for their small size, pleomorphism, and the presence of HAP. Like NB, serum granulations can seed particles upon transfer to serum-free medium, and their main protein constituents include albumin, complement components 3 and 4A, fetuin-A, and apolipoproteins A1 and B100, as well as other calcium and apatite binding proteins found in the serum. However, these serum mineralo-protein complexes are formed from the direct chemical binding of inorganic and organic phases, bypassing the need for any biological processes, including the long cultivation in cell culture conditions deemed necessary for the demonstration of NB. Thus, these serum granulations may result from physiologically inherent processes that become amplified with calcium phosphate loading or when subjected to culturing in medium. They may be viewed as simple mineralo-protein complexes formed from the deployment of calcification-inhibitory pathways used by the body to cope with excess calcium phosphate so as to prevent unwarranted calcification. Rather than representing novel pathophysiological mechanisms or exotic lifeforms, these results indicate that the entities described earlier as NB most likely originate from calcium and apatite binding factors in the serum, presumably calcification inhibitors, that upon saturation, form seeds for HAP deposition and growth. These calcium granulations are similar to those found in organisms throughout nature and may represent the products of more general calcium regulation pathways involved in the control of calcium storage, retrieval, tissue deposition, and disposal.

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Characterization of Granulations of Calcium and Apatite in Serum as Pleomorphic Mineralo-Protein Complexes and as Precursors of Putative Nanobacteria

Serum-derived granulations and purported nanobacteria (NB) are pleomorphic apatite structures shown to resemble calcium granules widely distributed in nature. They appear to be assembled through a dual inhibitory-seeding mechanism involving proteinaceous factors, as determined by protease (trypsin and chymotrypsin) and heat inactivation studies. When inoculated into cell culture medium, the purified proteins fetuin-A and albumin fail to induce mineralization, but they will readily combine with exogenously added calcium and phosphate, even in submillimolar amounts, to form complexes that will undergo morphological transitions from nanoparticles to spindles, films, and aggregates. As a mineralization inhibitor, fetuin-A is much more potent than albumin, and it will only seed particles at higher mineral-to-protein concentrations. Both proteins display a bell-shaped, dose-dependent relationship, indicative of the same dual inhibitory-seeding mechanism seen with whole serum. As ascertained by both seeding experiments and gel electrophoresis, fetuin-A is not only more dominant but it appears to compete avidly for nanoparticle binding at the expense of albumin. The nanoparticles formed in the presence of fetuin-A are smaller than their albumin counterparts, and they have a greater tendency to display a multilayered ring morphology. In comparison, the particles seeded by albumin appear mostly incomplete, with single walls. Chemically, spectroscopically, and morphologically, the protein-mineral particles resemble closely serum granules and NB. These particles are thus seen to undergo an amorphous to crystalline transformation, the kinetics and completeness of which depend on the protein-to-mineral ratios, with low ratios favoring faster conversion to crystals. Our results point to a dual inhibitory-seeding, de-repression model for the assembly of particles in supersaturated solutions like serum. The presence of proteins and other inhibitory factors tend to block apatite nuclei formation or to stabilize the nascent nuclei as amorphous or semi-crystalline spherical nanoparticles, until the same inhibitory influences are overwhelmed or derepressed, whereby the apatite nuclei grow in size to coalesce into crystalline spindles and films—a mechanism that may explain not only the formation of calcium granules in nature but also normal or ectopic calcification in the body.

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Fetuin-A/Albumin-Mineral Complexes Resembling Serum Calcium Granules and Putative Nanobacteria: Demonstration of a Dual Inhibition-Seeding Concept

Mineralo-organic nanoparticles form spontaneously in human body fluids when the concentrations of calcium and phosphate ions exceed saturation. We have shown previously that these mineralo-organic nanoparticles possess biomimetic properties and can reproduce the whole phenomenology of the so-called nanobacteria-mineralized entities initially described as the smallest microorganisms on earth. Here, we examine the possibility that various charged elements and ions may form mineral nanoparticles with similar properties in biological fluids. Remarkably, all the elements tested, including sodium, magnesium, aluminum, calcium, manganese, iron, cobalt, nickel, copper, zinc, strontium, and barium form mineralo-organic particles with bacteria-like morphologies and other complex shapes following precipitation with phosphate in body fluids. Upon formation, these mineralo-organic particles, which we term bions, invariably accumulate carbonate apatite during incubation in biological fluids; yet, the particles also incorporate additional elements and thus reflect the ionic milieu in which they form. Bions initially harbor an amorphous mineral phase that gradually converts to crystals in culture. Our results show that serum produces a dual inhibition-seeding effect on bion formation. Using a comprehensive proteomic analysis, we identify a wide range of proteins that bind to these mineral particles during incubation in medium containing serum. The two main binding proteins identified, albumin and fetuin-A, act as both inhibitors and seeders of bions in culture. Notably, bions possess several biomimetic properties, including the possibility to increase in size and number and to be sub-cultured in fresh culture medium. Based on these results, we propose that bions represent biological, mineralo-organic particles that may form in the body under both physiological and pathological homeostasis conditions. These mineralo-organic particles may be part of a physiological cycle that regulates the function, transport and disposal of elements and minerals in the human body.

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Bions: A Family of Biomimetic Mineralo-Organic Complexes Derived from Biological Fluids

Randall initially described calcified subepithelial papillary plaques, which he hypothesized as nidi for kidney stone formation. The discovery of calcifying nanoparticles (CNP) in many calcifying processes of human tissues has raised another hypothesis about their possible involvement in urinary stone formation. This research is the first attempt to investigate the potential association of these two hypotheses. We collected renal papilla and blood samples from 17 human patients who had undergone laparoscopic nephrectomy due to neoplasia. Immunohistochemical staining (IHS) was applied on the tissue samples using monoclonal antibody 8D10 (mAb) against CNP. Homogenized papillary tissues and serum samples were cultured for CNP. Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) analysis were performed on fixed papillary samples. Randall's plaques were visible on gross inspection in 11 out of 17 collected samples. IHS was positive for CNP antigen in 8 of these 11 visually positive samples, but in only 1 of the remaining 6 samples. SEM revealed spherical apatite formations in 14 samples, all of which had calcium and phosphate peaks detected by EDS analysis. From this study, there was some evidence of a link between the presence of Randall's plaques and the detection of CNP, also referred to as nanobacteria. Although causality was not demonstrated, these results suggest that further studies with negative control samples should be made to explore the etiology of Randall's plaque formation, thus leading to a better understanding of the pathogenesis of stone formation.

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Association between Randall's plaque and calcifying nanoparticles.

We retrospectively analyzed the records of patients requiring urinary diversion for intractable urinary incontinence from 1996 to 2011 at our institution. Patients were excluded from the cohort if they had evidence of bladder outlet obstruction or a cystectomy at time of diversion, or had been diverted for active malignancy. Sixty patients underwent a supravesical urinary diversion without concomitant cystectomy for intractable urinary incontinence. The etiology of the urinary incontinence was overtly neurogenic in 28 patients and non-neurogenic in 32 patients. Patients had an average follow-up of 45 months (range, 9-96). The indications for surgery, previous surgical interventions, complications of surgery, and long-term outcomes, including the incidence of pyocystis and the need for secondary cystectomy in the postoperative period were reviewed. RESULTS

Olivia Lee

Papers

Association between Randall's plaque and calcifying nanoparticles.

Reconstructive Urology Pyocystis After Urinary Diversion for Incontinence e Is a Concomitant Cystectomy Necessary