The role of calcifying nanoparticles in biology and medicine

Figures & data

Table 1 Properties of CNPs

Properties of calcifying nanoparticles
Morphological properties	80–500 nm in diameter (can pass through 100 nm filters).^Citation1,Citation16 Usually have coccoid, coccobacillar, or bacillar form, mineralized “igloo-like” structures.^Citation1,Citation16 Have hydroxyapatite shell, cellular-membranous structure, and central cavity.^Citation1,Citation16 Can form microscopic colonies (>1 mm in diameter) under low nutrient concentrations in the environment.^Citation1,Citation16 Divide by binary fission, fragmentation, and gemmation.^Citation1,Citation16 Can form thermoresistant biofilms^Citation1,Citation16
Tinctorial properties	Gram-negative; can be stained by DNA-specific dyes^Citation16^–^Citation24 (Hoechst 33258 at a concentration of 5 μg/mL for 50 minutes, propidium iodide, PicoGreen – they are specific after the filtration through 0.10–0.22 μm pores, staining after demineralization is optimal); can be revealed by von Kossa staining;^Citation16 staining by 2% uranyl acetate (possibly with lead citrate) can detect specific mucus on the hydroxyapatite shell;^Citation1 in the mineralized state can be stained by alizarin red S;^Citation26 can be stained by phosphotungstic acid^Citation16
Resistance and sensitivity	Resistant to 90°C heating for 1 hour.^Citation1 Resistant to γ-irradiation up to 30 kGray.^Citation1,Citation16 Resistant to 5% NaCl solution.^Citation1 In the mineralized state are resistant to lysozyme, proteinase K, certain other proteinases, lipases, amylases, alkali, ultrasound, X-ray, detergents, and solvents.^Citation1 Temperatures under 37°C suppress replication and prevent biofilm formation.^Citation1 Resistant to a wide spectrum of antimicrobial therapeutics:^Citation30 aminoglycosides (in pharmacological concentrations), chloramphenicol, lincosamides, cephalosporins, macrolides, fluoroquinolones, glycopeptides (in pharmacological concentrations), polymyxins, antituberculous agents, aminocyclitol, spectinomycin. Sensitive to tetracyclines,^Citation30 ampicillin,^Citation30 trimethoprim,^Citation30 trimethoprim-sulfamethoxazole,^Citation30 nitrofurantoin,^Citation30 5-fluorouracil,^Citation30 cytosine arabinoside,^Citation30 antimycin A,^Citation22 sodium azide,^Citation22 potassium cyanide,^Citation22 bisphosphonates (etidronate, clodronate),^Citation30 6-aminocaproic acid;^Citation30 in-vitro growth can be inhibited by EDTA, EGTA, and citrate^Citation22
Culture properties	Doubling time is 3 days; in media without serum, 6 days.^Citation1,Citation16 Passage can be performed in DMEM or RPMI-1640 independently of the presence of serum.^Citation1,Citation16 Optimal atmosphere for growth should contain 5% CO2 and 95% air.^Citation1,Citation16 Sensitive to β-mercaptoethanol, which stimulates growth of anaerobes, but cannot be cultivated under strictly anaerobic conditions.^Citation22 Calcify when serum concentration in the culture media is lower than 5%.^Citation1 Cytotoxic for fibroblasts and lymphocytes^Citation16
Biochemical properties	Metabolism is 10,000 times slower than in Escherichia coli.^Citation1 Incorporate uridine (into the expected nucleic acid),^Citation16,Citation21 methionine^Citation16 and aspartic acid^Citation25 (into the expected system of protein biosynthesis). Calcify under physiological pH (7.4).^Citation1,Citation16 Urease negative^Citation16
Detection methods	Bacterioscopic (DNA-specific dyes Hoechst 33258, propidium iodide, PicoGreen, staining after a demineralization is optimal) using scanning and transmission electron microscopy,^Citation16^–^Citation24 von Kossa staining, which is specific to calcium compounds,^Citation16 staining by 2% uranyl acetate (possibly with lead citrate) to detect specific mucus on the hydroxyapatite shell,^Citation1 staining by alizarin red S in the mineralized state,^Citation26 staining by phosphotungstic acid,^Citation16 after the long-term cultivation light microscopy with von Kossa staining is possible to be used for detection.^Citation1,Citation16 Bacteriological (cultivation in DMEM or RPMI-1640 without serum under 37°C for 4–6 weeks after filtration through 0.10–0.22 μm pores),^Citation1,Citation16 replication can be assessed by spectrophotometry (650 nm wavelength).^Citation16 Serological (CNP antigens and anti-CNP monoclonal antibodies 8/0 [to porin],^Citation17,Citation20 5/2 [to peptidoglycan],^Citation17,Citation20 and 8D10 [to porin]^Citation21,Citation32,Citation43 of NanoBiotech Pharma, Tampa, FL) – ELISA,^Citation43 immunohistochemistry,^Citation32 immunofluorescence reaction,^Citation32 immunoblotting, Ouchterlony immunodiffusion.^Citation20 Genomic (PCR), but there is doubt that existing primers are obtained on the basis of CNP nucleic acids, and not on the basis of nucleic acids of contaminating bacteria.^Citation2 Proteomic (sodium dodecyl sulfate polyacrylamide gel electrophoresis with further identification of protein bands by mass spectrometry)^Citation22
Methods of treatment of CNP-associated diseases	comET-therapy (tetracycline, EDTA, and mixture of nutrients)^Citation36,Citation41

Abbreviations: CNP, calcifying nanoparticles; DMEM, Dulbecco’s modified Eagle’s medium; EDTA, ethylenediaminetetraacetic acid; EGTA, ethyleneglycoltetraacetic acid; ELISA, enzyme-linked immunosorbent assay; PCR, polymerase chain reaction; RPMI-1640, Roswell Park Memorial Institute 1640 medium.

Table 2 Arguments supporting and contradicting the hypothesis that CNPs are nanobacteria, a unique form of life on Earth

Arguments in favor of theory that CNPs are living organisms (nanobacteria)	Arguments in favor of theory that CNPs are a physicochemical phenomenon (mineralo-protein complexes)
Morphological similarity to bacteria, cultivation on various media, binary fission^Citation16 Staining by DNA-specific dyes (Hoehst 33258, propidium iodide, PicoGreen)^Citation16^–^Citation24 Presence of DNA and RNA in ratio characteristic for Escherichia coli^Citation19,Citation22 Identification of DNA and RNA by electron microscopy^Citation49	Extremely small size (often smaller than 140 nm – presumably minimum possible size limit of life forms) Absence of fairly accurately sequenced genome
Incorporation of [^35S]methionine,^Citation16 [^3H]L-aspartic acid,^Citation25 and [5-^3H]uridine during cultivation^Citation16,Citation21	Association of hydroxyapatite of CNPs with albumin,^Citation6,Citation8–Citation10 fetuin-A,^Citation5,Citation9,Citation10 apolipoproteins,^Citation9,Citation10 proteins of coagulation and anticoagulation systems,^Citation9,Citation10 immunoglobulins,^Citation9,Citation10 macroglobulins,^Citation9,Citation10 complement proteins,^Citation9,Citation10 precursors of interleukins,^Citation10 kininogen,^Citation9,Citation10 fibronectin,^Citation9,Citation10 vitronectin,^Citation9,Citation10 vitamin D-binding protein,^Citation9,Citation10 precursor of antitrypsin,^Citation9,Citation10 serotransferrin,^Citation10 syntrophin,^Citation9,Citation10 Tamm-Horsfall glycoprotein,^Citation10 glycogenin,^Citation10 serum component of amyloid,^Citation10 salivary amylase,^Citation10 cytoskeleton proteins,^Citation9 adenosylhomocysteinase,^Citation9 hemoglobin subunits^Citation9 and other proteins
The presence of endotoxin and cross-reactivity with lipopolysaccharide of Chlamydia spp. and with antigens of Bartonella spp^Citation18	Cross-reactivity of MoAbs to presumably specific CNP proteins with serum proteins – albumin^Citation6,Citation8–Citation10 and fetuin-A^Citation5,Citation9,Citation10
Susceptibility to a number of antimicrobial chemotherapeutics possessing antimetabolic activity and not possessing the chelating activity,^Citation30 (nitrofurantoin, trimethoprim, trimethoprim-sulfamethoxasole), to 5-fluorouracil, cytosine arabinoside, 6-aminocaproic acid,^Citation30 antimycin A, sodium azide, potassium cyanide^Citation22	Morphological similarity to various mineral and mineralo-protein complexes containing inorganic calcium, phosphorus, and apatite compounds, and also proteins and phospholipids^Citation2,Citation6,Citation8–Citation10
Acceleration of replication and reduction of formation of biofilms after photobiostimulation^Citation29	Resistance to DNase and RNase activity^Citation5
Presence of proteins, including bacterial ones, after a demineralization^Citation16,Citation20,Citation21 (EF-Tu,^Citation22,Citation26 EF-G,^Citation26 GroEL,^Citation22 dihydrolipoamide acetyltransferase of pyruvate dehydrogenase,^Citation22 polyribonucleotide nucleotidyl transferase,^Citation22 fructose binding aldolase,^Citation26 chaperone C1pB^Citation26)	Critical influence of inert gas, CO2, and NaHCO3 concentration on CNP replication^Citation6
Stimulation of replication by β-mercaptoethanol and absence of replication under strictly anaerobic conditions^Citation22	CNP formation under physiological serum conditions after certain physiological alterations of homeostasis, which is not characteristic of living organisms^Citation5^–^Citation10
Pathogenic activity of demineralized CNPs^Citation31 and distinct pathogenicity of CNPs and hydroxyapatite crystals^Citation23

Abbreviations: CNP, calcifying nanoparticle; MoAb, monoclonal antibody; EF-Tu, elongation factor thermo unstable; EF-G, elongation factor G.

Table 3 The relation of CNPs to various diseases

Study	Disease or pathological condition	Method of detection of association between CNPs and disease or pathological condition
Kajander and Ciftçioglu^Citation16	Nephrolithiasis	Serological, bacterioscopic, bacteriological
Ciftcioglu et al^Citation17	Nephrolithiasis	Serological, bacterioscopic, bacteriological
Hjelle et al^Citation18	Polycystic kidney disease	Serological, bacterioscopic, bacteriological
Sedivy and Battistutti^Citation46	Formation of psammoma bodies in ovarian malignant tumors	Serological
Hudelist et al^Citation19	Formation of psammoma bodies in ovarian malignant tumors	Serological
Khullar et al^Citation20	Nephrolithiasis	Serological, bacterioscopic, bacteriological
Maniscalco et al^Citation41	Coronary artery calcification	Serological
Miller et al^Citation21	Calcification of coronary arteries and cardiac valves	Serological, bacterioscopic, bacteriological
Pretorius et al^Citation48	HIV infection	Serological
Puskás et al^Citation25	Atherosclerotic plaques	Serological, bacterioscopic, bacteriological
Shoskes et al^Citation36	Type III chronic prostatitis/chronic pelvic pain syndrome	Serological
Wen et al^Citation47	Cholecystolithiasis	Serological, bacterioscopic, bacteriological
Kumar et al^Citation22	Randall’s plaques	Serological, bacterioscopic, bacteriological
Shiekh et al^Citation26	Renal tubular calcification	Bacteriological (animal model)
Wang et al^Citation23	Black pigment gallstones	Serological, bacterioscopic, bacteriological (animal model)
Agababov et al^Citation49	Pathological placental calcification	Bacterioscopic
Bratoz-Perez et al^Citation42	Aortic valves calcification	Bacterioscopic, bacteriological
Ciftcioglu et al^Citation32	Randall’s plaques	Serological, bacterioscopic, bacteriological
Schwartz et al^Citation31	Calcification of arteries with damaged endothelium	Bacterioscopic, bacteriological (animal model)
Zhou et al^Citation37	Type III prostatitis	Serological, bacterioscopic, bacteriological
Candemir et al^Citation43	Mitral annular calcification	Serological
Chen et al^Citation33	Nephrolithiasis	Serological, bacterioscopic, bacteriological
Hu et al^Citation44	Calcification of cardiac valves during rheumatic heart disease	Serological, bacterioscopic, bacteriological
Hu et al^Citation34	Nephrolithiasis	Bacterioscopic, bacteriological (animal model)
Shen et al^Citation38	Type III prostatitis	Bacterioscopic, bacteriological (animal model)
Zhang et al^Citation39	Interstitial cystitis/painful bladder syndrome	Serological, bacterioscopic, bacteriological
Zhang et al^Citation40	Testicular microlithiasis	Serological, bacterioscopic, bacteriological
Tulunay Kaya et al^Citation45	Coronary artery calcification	Serological

Abbreviations: CNP, calcifying nanoparticle; HIV, human immunodeficiency virus.

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