Suffocation of Nerve Fibers by Living Nanovesicles: A Model Simulation Andrei P. Sommer Central Institute of Biomedical Engineering, University of Ulm, 89081 Ulm, Germany Received December 3, 2003
A model using nanospheres to allow the simulation of the nonspecific interaction of nanobacteria (NB), one with another or with body tissues, is established. Depending primarily on their concentrations and stress levels, these apatite nanovesicles may nucleate thrombogenic conglomerates in blood, or self-assemble to dense nanoclay layers on surfaces in the body. Partial or total encapsulation of nerve fiber bundles by such mineral layers may interrupt the metabolic exchanges between the surrounded tissue and its immediate environment and may restrict signaling processes. The presented model could provide detailed insight into plaque formation triggered by NB, and the parameters encouraging it. Keywords: nanovesicles • nanoclay • self-assembly • perineurium • peripheral neuropathy • HIV
Introduction In a recent study, a link was established between nanobacteria (NB) and HIV.1 The presence of NB in HIV-infected patients is indicated by the interrelation of seven experimental findings: (1) The perineurium was virtually coated with apatite in many of the diabetic patients afflicted with peripheral neuropathy.2,3 (2) NB are protected by a porous mineral shell consisting of apatite.4 (3) Exposed to physiological or biomechanical stress, NB have been observed to produce in vitro a slime promoting rapid colony formation by attachment one to another and/or to surfacessa mechanism proposed to be instrumental in inducing various forms of pathogenic calcification.5 (4) HIV-infected patients presented extreme conditions of pathogenic calcification.6 (5) The situation of numerous HIVinfected patients is additionally burdened by peripheral neuropathysa disease frequently associated with diabetes mellitus. (6) Low-level light was found to compensate stress in cultured NB.7,8 (7) Peripheral neuropathy has been reported to dramatically ameliorate with low level light therapy.9 Modeling the Perineurium. It is thus motivating to analyze the interplay between NB and biological surfaces. Interestingly, the physiological effect is not the same when NB are deposited on an extended plane surface, as opposed to a cylindrical surface with a small diameter, which can easily be completely enveloped by the NB. Cylindrical surfaces could be tubular systems, e.g., blood vessels (internal deposition), or the perineurium (external deposition). Clearly, in both cases, a layer of NB may partially or totally inhibit metabolic processes by blocking the permeability of the tissue. For a better understanding of external deposition processes at curved surfaces, an experimental model using two cylindrical objects with curvatures mimicking relevant biological systems was used: a cleaved optical fiber (Ø ) 125 µm) consisting of quartz glass (Figure 1), and human hair (Ø ≈ 65 µm) shown in Figure 4. An 10.1021/pr034122c CCC: $27.50
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Figure 1. Light microscopy image showing a piece of an optical fiber (Ø ) 125 µm) on titanium disk. The brownish nanoclay around the fiber was formed by a drop of an aqueous suspension based on 60-nm polystyrene nanospheres, slowly evaporating on the disk. Initially, the fiber was covered by the drop, reaching until the right margin of the disk. The grayish segment of the fiber on the right was not coated with nanoclay.
evident difference between the hair and the optical fiber is, however, the surface topography. The surface of the fiber is smooth on the subnanoscale, conforming to the specifications for optical communication. The surface of the hair is rough, on the microscale, as well as on the nanoscale, as can be verified in Figure 4. Modeling Nanobacteria. NB isolated from mammalian blood have diameters reaching 80 to 300 nm. Because of their spherical architecture and the relatively narrow size distribution, it appears reasonable to interpret NB as simple nanospheres. Nanosuspensions recommend themselves for simulation of the interaction of NB with another in blood10 or with Journal of Proteome Research 2004, 3, 667-669
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Figure 2. (left) SEM image of the opposite end of the fiber presented in Figure 1, revealing unilateral contact with the suspension (whitish layer on upper half). Bar ) 10 µm. (right) SEM image of the zone within the selected window in the left image shows numerous isolated 60-nm nanospheres. Clearly visible are the cracks in the loosely packed nanoclay layer coating the fiber. Bar ) 300 nm. Fiber was removed from its original position (shown in Figure 1).
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been chosen as substrate material because of its hydrophilic nature, and because of the extensive experimental data on patterns formed by evaporation of nanosuspensions on this biomaterial.11-13 By placing the disks in closed Petri dishes, slow evaporation was ascertained. Slow evaporation is expected to extend the contact time between the nanospheres and the cylindrical samples, thus allowing the exaggeration of the simulated biological scenario. Prolonged contact-time between the NB suspended in body fluids and tissues seems realistic, in particular under spatial confinement, e.g., around the perineurial tube or in the kidneys.4 Using the glass fiber (hydrophilic) and the human hairs (hydrophobic), it was possible to evaluate the effect of polarity on attachment. The samples were examined by light microscopy and scanning electron microscopy (SEM).
Results
Figure 3. (left) Representative SEM image of piece in the middle of the fiber shown in Figure 1. Fiber is partially coated with dense nanoclay film consisting of 60-nm nanospheres. Bar ) 10 µm. (right) SEM image presents the situation within the frame marked in the image on the left. Bar ) 1 µm.
Figure 4. (left) SEM image of two human hairs on titanium disk. On both sides dried nanoclay consisting of 60-nm polystyrene nanospheres, formed by a slowly evaporating drop of an aqueous nanosuspension. Initially, both hairs were covered by the drop. Bar ) 10 µm. (right) Representative SEM image of a segment of one of the hairs presented in the image on the left, showing some 60-nm nanospheres (f arrow). Bar ) 1 µm. Hair was not washed for one week.
curved tissue surfaces within body fluids. Self-organization of nanospheres of the size of NB, deposited from drops of aqueous suspensions on both hydrophobic and hydrophilic surfaces, has been investigated now in detail.11,12 On planar hydrophilic surfaces (titanium and mica), slow evaporation of drops of aqueous suspensions, containing 60 and/or 200 nm polystyrene nanospheres, formed highly ordered, densely packed crystalline structures.13 Similar to nanospheres, NB may self-assemble on suitable surfaces.
Materials and Methods Two human hairs and the optical fiber were positioned on mirror-polished titanium disks. A drop of an aqueous suspension of about 10 µL, containing 60 nm nanospheres (Duke Scientific, Palo Alto, CA), was symmetrically placed via a syringe on top of the hairs and on the fiber, respectively. Titanium has 668
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Figure 1 shows a representative picture for the situation when a drop of the nanosuspension was put exactly on top of the glass fiber. Due to the hydrophilic character of the fiber, the suspension is symmetrically distributed. Nanoclay can be found on both sides of the fiber, parallel to its axis, forming an elongated ring, embedding the fiber at its ends. A closer inspection of the photograph reveals that the extension of the whitish layer on top of the fiber is limited to the piece which had contact with the nanosuspension. Figure 2 allows visualizing the situation on the nanoscale. The SEM image on the right exposes solitary nanospheres attached to the fiber and the structure of the whitish layer, consisting of densely packed nanoclay. Figure 3 exposes a dense nanoclay film at the middle of the fiber. Such films may be useful to simulate both the formation dynamics and the stability of NB-plaques, as indicated by missing film-piece, visible in the magnified SEM image on the right. Loosened NB-plaques are thought to possess a thrombogenic potential. Figure 4 shows a SEM image of two human hairs. Due to the hydrophobic character of the employed hairs, the drop is asymmetrically distributed around them. Although the hairs had a rough surface topography, only a few nanospheres could be identified on top of them.
Conclusions NB seem to have an ideal size for effective surface covering deposition by self-assembly, driven by energetically favorable conditions and the strongly hydrophilic nature of the apatite mineral. In vivo, the tendency to attach to tissue surfaces could be elevated by slime containing calcium and phosphate, produced by stressed NB. Massive physiological changes in the blood milieu, as for example, represented by the multitude of opportunistic infections described in HIV,14 may stimulate NB to slime production.10 Slime-assisted perineurial deposition of a large number of stressed NB, as presumably realized in peripheral neuropathy, could progressively cut signal transduction along the enclosed bundle of nerve fibers, primarily via metabolic isolation of the perineurium, and second by destroying the elasticity of the perineurial envelope, restricting the vital capability of the perineurium to stretch and deform. Methods allowing simulation of the interaction of the living nanovesicles with tissues, and the observed control of their slime secretion by light, may hold the key to further progress, and could inspire novel strategies preventing their attachment to surfaces.
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Suffocation of Nerve Fibers by Living Nanovesicles
References (1) Sommer, A. P. J. Proteome Res. 2003, 2, 665. (2) Kalimo, H.; Maki, J.; Paetau, A.; Haltia, M. Muscle Nerve 1981, 4, 228. (3) King, R. H.; Llewelyn, J. G.; Thomas, P. K.; Gilbey, S. G.; Watkins, P. J. Neuropathol. Appl. Neurobiol. 1988, 14, 105. (4) Kajander, E. O.; Ciftcioglu, N. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 8274. (5) Sommer, A. P.; Kajander, E. O. Cryst. Growth Des. 2002, 2, 563. (6) Nadler, R. B.; Rubenstein, J. N.; Eggener, S. E.; Loor, M. M.; Smith, N. D. J. Urol. 2003, 169, 475. (7) Sommer, A. P.; Hassinen, H. I.; Kajander, E. O. J. Clin. Laser Med. Surg. 2002, 20, 241.
(8) Sommer, A. P.; Oron, U.; Pretorius, A. M.; McKay, D. S.; Ciftcioglu, N.; Mester, A. R.; Kajander, E. O.; Whelan, H. T. J. Clin. Laser Med. Surg. 2003, 21, 229. (9) Sommer, A. P.; Oron, U.; Kajander, E. O.; Mester, A. R. J. Proteome Res. 2002, 1, 475. (10) Sommer, A. P.; Pretorius, A. M.; Kajander, E. O.; Oron, U. Cryst. Growth Des. 2004, 4, 45. (11) Sommer, A. P.; Franke, R. P. NanoLett. 2003, 3, 321. (12) Sommer, A. P.; Franke, R. P. NanoLett. 2003, 3, 573. (13) Sommer, A. P.; Ben-Moshe, M.; Magdassi, S. J. Phys. Chem. B 2004, 108, 8. (14) Klatt, E. C. Adv. Cardiol. 2003, 40, 23.
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