Identification of Nanobacteria in Human Arthritic Synovial Fluid by

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Environ. Sci. Technol. 2008, 42, 3324–3328

Identification of Nanobacteria in Human Arthritic Synovial Fluid by Method Validated in Human Blood and Urine using 200 nm Model Nanoparticles TOSHIYUKI TSURUMOTO,† DAN ZHU,‡ AND A N D R E I P . S O M M E R * ,‡ Department of Orthopaedics, Graduate School of Biomedical Sciences, Nagasaki University, 1-7-1, Sakamoto, Nagasaki, 852-8501, Japan, and Institute of Micro and Nanomaterials, University of Ulm, 89081 Ulm, Germany

Received November 13, 2007. Revised manuscript received January 28, 2008. Accepted January 30, 2008.

Earlier we introduced a biosensor for the identification of nanobacteria in water drops. Here, we generalize its principle and apply it to identify nanobacteria in synovial fluid from a patient with osteoarthritis. Results indicate the prevalence of nanobacteria in the synovial fluid. The identification method is applicable to body fluids such as unfiltered human blood and urine, is independent of culturing procedures, and permits for a rapid detection of nanoparticles in liquid drops. In view of increasing clinical evidence on a contribution of nanobacteria in disease, their reported detection in HIV-infected people in South Africa, laboratory experiments indicating the excretion of viable (i.e., propagating) nanobacteria from humans via urine, the use of human excreta in agricultural irrigation, models predicting an injection of nanoaerosols contained in irrigation water enriched with human excreta into the atmosphere, and the identification of nanobacteria in the terrestrial atmosphere, promote the identification method described in this work to an important tool to monitor nanobacteria in body fluids and environmental samples.

Introduction There is a growing interest to evaluate the uptake of nanoparticles by biosystems (1). This includes information on both their dwell time in the body and their rate of excretion. However, to monitor nanoparticles in body fluids is by no means trivial. In general, body fluids are rich in a variety of minerals, biomacromolecules, and adhesive components, specifically glycoproteins. Depending on their concentration and chemical affinity, these constituents tend to mask the nanoparticles, particularly under stationary conditions. This is the principal obstacle in recognizing nanoparticles in extracted samples of body fluids. Theoretically, the simplest way of monitoring them in drops of fluids is to discriminate them by their typical size and shape. Practically, this problem has been solved for drops of aqueous nanosuspensions containing two differently sized nanospheres (2). We note that the actual interest in monitoring nanoparticles in body fluids is not limited to synthetic systems (nanoscale drug * Corresponding author e-mail: [email protected]. † Nagasaki University. ‡ University of Ulm. 3324

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carriers, nanoparticles in cosmetics and pollution particles) but includes a large body of nanoscale objects of biological origin, for instance, viruses and nanobacteria (NB). From the perspective of their size (predominantly 60–300 nm), spherical shape and a mineral shell consisting of apatite, NB are intrinsic natural nanoparticles. Their spreadsNB have now been identified in humans on four continentssand increasing evidence for their implication in disease, including but not limited to kidney stones (3), heart disease (4–6), and HIV (7–9) are sufficient motivation to focus on practical methods allowing for their identification in body fluids. Noting the morphological parallels between NB and synthetic nanospheres, on the one hand, and that NB prevail in the blood of infected humans and are eliminated from the body via urine, on the other hand, model systems allowing us to systematically monitor nanoparticles in these body fluids are of special interest. Previously, we introduced a biosensor and a concept allowing one to identify NB in a sea of equal sized nanoparticles originating from pollution sources (10). Here, we generalize the concept and apply it to identify nanoparticles in three body fluids: blood, urine, and synovial fluid (SF). The identification method is independent of culturing, and permits for the rapid detection of nanoparticles in aqueous drops. The method exploits the interplay between convection, carrying the material suspended in an evaporating sessile drop to the periphery of the drop, and anchoring of the material to the substrate. This principle propelled the development of an improved model (11) which was recently confirmed for molecules and carbon nanotubes (12–14). Its parameters are summarized in a self-explanatory model (Figure 1). Earlier we reported the potential identification of NB in SF of knee joints of six patients (four with rheumatoid arthritis, two with osteoarthritis) (17). In the previous study the SF was kept for two months under mammalian cell culture conditions before NB-like particles could be imaged in the liquid. The possible implication of nanoparticles in general and NB in particular in joint disease justifies their inclusion into the catalogue of abnormalities of inflammatory SF: Crystalline forms of calcium phosphate are prevalent in SF from patients with osteoarthritis and are identifiable via wet drop analysis by polarized light microscopy (18). Cholesterol crystals are occasionally present in SF. In the light microscope they appear as rhomboid plates and are birefringent (19). It is believed that their large size prevents them from inducing joint inflammation, and that synovial proteins play a role in their formation (20). SF from the normal knee joint is optically

FIGURE 1. Parameters controlling the organization of nanoparticles in a water drop: 1. convection, 2. nanoparticle-substrate interaction, 3. nanoparticle-nanoparticle interaction, 4. subaquatic water layer, 5. marginal outflow, 6. temperature gradient ∆T, 7. gravity Fg, 8. nanoparticle properties, 9. substrate structure and 10. evaporation time. The interplay of the independent parameters 1 and 2 determines if a ring or a uniform deposition pattern is formed (15). (Image from ref 15). It is worth noting that the interaction spectrum described by Figure 1 decreases with increasing particle size, for instance, pollen grains tend to stick upon precipitation to the central area of substrate (16). 10.1021/es702857s CCC: $40.75

 2008 American Chemical Society

Published on Web 04/02/2008

FIGURE 2. Top left: Light microscopy photograph of dried ring formed by slowly evaporating blood drop on polystyrene Petri dish. Sodium citrate 3.13% was supplemented to prevent clotting before dilution 1:10 with ultra pure water. Top right: SEM image of the ring periphery shows 200 nm nanospheres (arrow). Bottom left: Light microscopy photograph of dried pattern from drop of urine diluted 1:10 with ultra pure water on same substrate. Bottom right: SEM image of edge of the pattern shows an urinary crystal; at its tip are clusters of 200 nm nanospheres.

FIGURE 3. Light microscopy photographs show deposition patterns formed by fast evaporation of drops of blood (left) and urine (right), both diluted 1:2 with ultra pure water. clear. In inflammatory joints it is turbid. In patients with osteoarthritis turbidity was related to the degree of the joint inflammation, and traced back to the cellularity of the fluid (21). Further constituents of arthritic SF are cell-derived microparticles (22, 23). Those isolated from healthy volunteers had irregular contour lines (A. Sommer, personal communication with R. Berckmans).

Materials and Methods Figure 2 is a comprehensive synopsis illustrating the application of the model shown in Figure 1 for the identification

of 200 nm polystyrene nanoparticles (Duke Scientific, Palo Alto, CA) in blood and urine, both diluted 1:10 with ultra pure water. As can be seen, a blood drop (10 µL) forms during an evaporation time of 26 h (in closed Petri dish) at 24 °C a perfect ring on the dish. Under identical conditions, a drop of urine of the same size forms a uniform deposition pattern. Preferentially, 200 nm nanoparticles contained in the fluids could be clearly identified around the periphery of the deposition patterns. Figure 3 has been included to demonstrate the dependence of the patterns on the time of evaporation and dilution (cf. caption to Figure 2). Normally, VOL. 42, NO. 9, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. Left: Photograph of 20 µL drops of SF diluted 1:10 in ultra pure water on 35 mm Petri dish. Right: Light microscopy photograph of dried ring pattern formed by one drop after a time of evaporation of 7 days in closed dish. The arrow marks the position of the area shown in Figure 5. Diameter of the ring ) 4.9 mm.

FIGURE 5. Left: Light microscopy photograph of marked area in Figure 4 shows a multitude of dark dots in dried synovial fluid. Bar ) 10 µm. Right: SEM image reveals a large number of spherical nanoparticles in the corresponding part of the ring. Bar ) 1 µm. little diluted drops evaporating in air (at relative humidity levels of 30%, evaporation time 1 h) form compact patterns. Identification of nanoparticles in patterns similar to those presented in Figure 3 is virtually impossible. The reason for this is simple: during slow evaporation, suspended material contained in the drops is transported by the convection to the edge of the drop, where it can be identified by standard high resolution imaging methods. When the time of evaporation is too short, the carrier liquid, which transports the suspended material to the periphery of the drop, vanishes before the material has reached the periphery. As a consequence, the suspended material remains buried under a thick layer of lighter components, and is invisible for the applied imaging methods.

Results In our first paper reporting on the possible presence of NB in the SF of patients with rheumatoid arthritis and osteoarthritis, the NB-like particles could first be imaged by light microscopy after a consecutive period of two months of culturing in cell culture medium (17). Culturing was performed according to the protocol described in the literature (3). In this study we used SF from the knee joint of a 71 year-old patient with osteoarthritis. Figure 4 (left) shows three polystyrene Petri dishes, one of which contains three 20 µL drops of SF, diluted 1:10 in ultra pure water, passed through a filter of pore size 0.22 µm. The dish was sealed with a permeable tape and placed together with two other dishes 3326

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(containing water) into a larger Petri dish, which was closed to extend the time of evaporation. The ring pattern in Figure 4 (right) formed in seven days, during evaporation at room temperature. The representative light microscopy image of an area proximal to the periphery of a SF ring (Figure 5, left), marked by an arrow in Figure 4 (right), shows a multitude of dark dots. Figure 5 (right) is a scanning electron microscopy (SEM) image of the same area and displays a large number of spherical nanoparticles. The SEM image in Figure 6 depicts numerous 100–300 nm nanoparticles in a peripheral field, at higher resolution. They are surrounded by a mucous matrix, a biomarker for NB. The corresponding energy dispersive spectroscopy (EDAX) image (Figure 7) documents the presence of calcium, phosphorus and oxygen. Calcium phosphate is, together with the shape, size range, and mucous matrix, the typical fingerprint of NB. Subsequent to the extraction from the knee joint the SF was centrifuged (30 min at 3000 rpm). The practically cell-free supernatant was then stored in a deep freezer at -30 °C.

Discussion A potential equivalence of the 100–300 nm objects shown in Figure 6 to the microscopic calcium phosphate crystals, recurrently identified in osteoarthritic SF, can be safely excluded because of their characteristic size, ranging from 0.49 to 17.9 µm (25). Diseases caused by basic calcium phosphate crystals in synovial fluid occur frequently in

FIGURE 6. SEM image of ring periphery displays spherical objects, predominantly in the size range 100–300 nm.

FIGURE 7. EDAX performed at two spots marked in Figure 6, with evidence for calcium, phosphorus, and oxygen. The presence of sodium and magnesium in NB has been reported by others (24). osteoarthritic joints (26). Such crystals can be identified by light microscopy (27). Thus, it is reasonable to assume a similar implication of NB in joint disease in general and osteoarthritis in particular. There are uncertainties in our

apparently straightforward experiments, which should be clearly mentioned: Presently, we do not know whether NB in SF are unique to arthritic joints or not. Clarification of this critical point would involve the analysis of SF of healthy controls. However, the SF volume in the knee joints of healthy people is so small that extraction, even of minimal amounts, is not justifiable on an ethical basis. A major uncertainty, also reflected in the current literature, concerns the viability of heavily mineralized NB. One principal problem in NB research is that the material employed for the analysis of NB stems from samples grown under cell culture conditions. NB integrated in tissues present usually a thick apatite shell, which prevents their proper analysis. In addition, separation and isolation of tissue-NB is experimentally most compelling and has never been performed satisfactorily. Prerequisite for getting insight into the core of the nanovesicles and the nature and quantity of the associated proteins, are samples containing a substantial amount of minimally mineralized NB, for instance SF. The relatively smooth shape of the NB found in SF suggests indeed a low degree of mineralization (NB grown under cell culture condition and those identified in high perfusion organs, i.e., kidneys and heart, frequently present an irregular shape). Because of the emergence of the nanoparticles in systems as different as SF diluted with ultra pure water (changing concentration), and SF diluted with cell culture medium (constant concentration) (17), it is reasonable to assume that they were not generated ex vivo in the course of a concentration depended process. Recent trials to identify nucleic acids in mineralized NB remained fruitless, either because they did not contain nucleic acids, or because of the use of extensively mineralized NB (28). As we pointed out earlier, slime is a biomarker for NB (10). Its involvement in immune reactions provoked by NB (29) seems possible. Focus on the presence (or absence) (30) of this characteristic, as well as a strict restriction to facts (31), are necessary for progress in future work. NB were probably first observed in a relationship to peripheral neuropathy (32–35), and in SF (36–38) of patients with joint disease. Present research focuses exclusively on the chemical characterization of the mineral shells of NB and on the analysis of the biological content in their interior. For future research we recommend another route: Instead of attempting to probe a genetic signature in the interior of mineralized NB, it could be more appropriate to focus on the proteins collected and/or secreted by NB. We demonstrated the identification of nanoparticles in blood and urine, and identified NB in SF in a way that their native environment is preserved. The methodology applied by us allows one to circumvent culturing procedures and is quick, i.e., by using smaller drops. This should encourage competent groups to apply our method to detect relevant nanoparticles in body fluids, and isolate NB and proteins possibly secreted by them. A method to identify nanoparticles in liquids in general, and in body fluids in particular, has been introduced and validated by the use of 200 nm nanospheres in blood and urine, and successfully applied for the first time for NB in SF. The method recommends itself for the screening of body fluids for NB on a large scale, as could be necessary for the assessment of coinfections with NB in HIV-infected people in sub-Saharan Africa (7). Considering the reported identification of NB in the terrestrial atmosphere on the one hand (39, 40), and the probability of their massive injection into the atmosphere via agricultural use of water enriched with human urine containing potentially viable NB for irrigation, in particular spray irrigation, and human (and animal) excreta containing potentially viable NB for crop fertilization (41, 42), on the other hand, simple methods facilitating their identification in open water reservoirs and in environmental samples such as rain or ice particles (43) should be welcomed. The recently reported identification of VOL. 42, NO. 9, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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NB in 48 of 74 heart valves, supports the possibility that NB are not simply innocent bystanders but play an active role in heart valve calcification (6), and is expected to prompt additional study. In view of the morphological parallels between mineralized NB and synthetic nanoparticles, including carbonaceous pollution nanoparticles, a systematic application of the identification technique established in this work could help to set up a precise map of the circulation and distribution lines of nanoparticles in the body and to get valuable insights into the routes of their excretion. The technique promises to become a sensitive and powerful detection tool for all kind of nanoparticles in all body fluids, a “PCR for nanoparticles”.

(19) (20) (21) (22)

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Acknowledgments We thank Takashi Suematsu, Electron Microscope Center, Graduate School of Biomedical Sciences, Nagasaki University, for performing the SEM work.

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