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Superior Biolubricant from a Species of Red Microalga Shoshana (Malis) Arad,*,† Lev Rapoport,‡ Alex Moshkovich,‡ Dorit van Moppes,† Mark Karpasas,† Roxana Golan,§ and Yuval Golan| Department of Biotechnology Engineering, Ben-Gurion UniVersity of the NegeV, P.O. Box 653, Beer-SheVa 84105, Israel, Tribology Laboratory, Holon Institute of Technology, Holon 58102, Israel, Department of Chemical Engineering, Ben-Gurion UniVersity of the NegeV, Beer-SheVa 84105, Israel, and Department of Materials Engineering and Ilse Katz Nanotechnology Center, Ben-Gurion UniVersity of the NegeV, Beer-SheVa 84105, Israel ReceiVed March 5, 2006. In Final Form: May 25, 2006 The rheological properties of the sulfated polysaccharide of the red microalga Porphyridium sp., a heteropolymer with a molecular weight of 3-5 × 106 Da, indicated that this material might be an excellent candidate for lubrication applications: the viscosity of the polysaccharide is stable over a range of temperatures, pH values, and salinities. In this study, various rheological and lubricant properties of the polysaccharide were evaluated in comparison with those of a widely used biolubricant, hyaluronic acid. The viscosity of the Porphyridium sp. polysaccharide remained essentially unchanged in a temperature range of 25-70 °C. In tribology tests on a ball-on-flat ceramic pair, the values for the friction coefficient and wear rate for the pair lubricated with polysaccharide were remarkably lower than those for hyaluronic acid, especially at high loads. In a test on a steel ring/ultrahigh-molecular-weight polyethylene (UHMWPE) block pair, the wear tracks on the surface of the UHMWPE were more pronounced for hyaluronic acid than for the polysaccharide. Atomic force microscopy showed that the polysaccharide was effectively adsorbed onto mica surfaces, forming ultrathin coating layers in the nanometer range. As is required for biolubricant applications, the polysaccharide was not degraded by hyaluronidase. The stability of the Porphyridium sp. polysaccharide to heat and to hyaluronidase combined with its ability to reduce friction and wear indicate its potential as an advantageous biolubricant.
Introduction One of the fields of study currently at the forefront of tribology research is that of dealing with the friction, wear, and lubrication of biomaterials, i.e., hydrogel lubricants with low friction, such as hyaluronic acid, that are currently in high demand for biomedical applications.1 Such polysaccharides find application as lubricants for the amelioration of cartilage friction in joints. They are also used as lubricants for contact lenses, catheters, and artificial oesophagi. Although it is generally accepted that hyaluronic acid is an effective lubricant, recent studies have shown that under physiological conditions adhesion of this mucopolysaccharide to surfaces is very weak.2 As a result, the initially low friction forces between surfaces lubricated with hyaluronic acid increase rapidly with sliding time.3,4 As a potential substitute for hyaluronic acid in medical/cosmetic applications, we evaluated the sulfated polysaccharide obtained from the red microalga Porphyridium sp., since rheological studies indicated that it would have good lubrication properties.5 Porphyridium sp. cells are encapsulated within a sulfated polysaccharide in the form of a gel. The external part of the polysaccharide dissolves in the medium, and thus the viscosity * To whom correspondence should be addressed. E-mail: arad@ bgu.ac.il. Phone: 972-547-772809. Fax: 972-8-6479067. † Department of Biotechnology Engineering, Ben-Gurion University of the Negev. ‡ Holon Institute of Technology. § Department of Chemical Engineering, Ben-Gurion University of the Negev. | Department of Materials Engineering and Ilse Katz Nanotechnology Center, Ben-Gurion University of the Negev. (1) Gong, J. P.; Osada, Y. Prog. Polym. Sci. 2002, 27, 3-38. (2) Tadmor, R.; Chen, N. H.; Israelachvili, J. N. J. Biomed. Mater. Res. 2002, 61 (4), 514-523. (3) Tadmor, R.; Chen, N. H.; Israelachvili, J. N. Biophys. J. 2002, 82 (1), 799. (4) Tadmor, R.; Chen, N. H.; Israelachvili, J. N. Macromolecules 2003, 36, (25), 9519-9526. (5) Arad (Malis), S.; Weinstein, J. Biomedic (Israel) 2003, 1, 32-37.
of the medium is increased.6,7 The polysaccharide is a ∼3-5 × 106 Da heteropolymer, with about 7% sulfate on a dry weight basis.8-13 The main sugars of the polysaccharide are xylose, glucose, and galactose, in various ratios; some minor sugars are also present. The polysaccharide is negatively charged due to the presence of glucuronic acid and half-sulfate ester groups and contains a noncovalently bound 66 kDa glycoprotein.14 A disaccharide building block, aldobiuronic acid [3-O-(R-Dglucopyranosyluronic acid)-L-galactopyranose],15 has been characterized.16 In concentrated solutions, the viscosity of the polysaccharide is stable over a wide range of pH values (2-9), temperatures (30-120 °C), and salinities.10 It has been suggested that the biopolymer chain molecules adopt an ordered conformation in solution and that the polysaccharide takes the form of a double or triple helix.17 The stiffness of the polysaccharide is in the same range as that of xanthan and DNA.18 (6) Ramus, J. J. Phycol. 1972, 8, 97-111. (7) Ramus, J. In Algal Biomass Technologies; Barclay, W. R., McIntosh, R. P., Eds.; J. Cramer: Berlin, 1986; p 51-55. (8) Heaney-Kieras, J.; Roden, L.; Chapman, D. J. Biochemistry 1977, 165, 1-9. (9) Medcalf, D. G.; Scott, J. R.; Brannon, J. H.; Hemerick, G. A.; Cunningham, R. L.; Chessen, J. H.; Shah, J. Carbohydr. Res. 1975, 44, 87-96. (10) Geresh, S.; Arad (Malis), S. Biores. Technol. 1991, 38, 195-201. (11) Arad (Malis), S. In Chemicals from Microalgae; Cohen, Z., Ed.; Taylor & Francis: London, 1999; pp 282-291. (12) Heaney-Kieras, J.; Kieras, J. F.; Bowen, D. V. Biochem. J. 1976, 155, 181-18. (13) Arad (Malis), S. In Algal Biotechnology; Stadler, T., Mollion, J., Verdus, M. C., Karamanos, Y., Morvan, H., Christiaen, D., Eds.; Elsevier Applied Science: London, 1988; pp 65-87. (14) Shrestha, R. P.; Weinstein, Y.; Bar-Zvi, D.; Arad (Malis), S. J. Phycol. 2004, 40, 568-580. (15) Geresh, S.; Dubinsky, O.; Arad (Malis), S.; Christiaen, D.; Glaser, R. Carbhydr. Res. 1990, 208, 301-305. (16) Heaney-Kieras, J.; Chapman, D. J. Carbohydr. Res. 1976, 52, 169-177. (17) Eteshola, E.; Karpasas, M.; Arad (Malis), S.; Gottlieb, M. Acta Polym. 1998, 49, 549-556. (18) Eteshola, E.; Gottlieb, M.; Arad (Malis), S. Chem. Eng. Sci. 1996, 51, 1487-1494.
10.1021/la060600x CCC: $33.50 © 2006 American Chemical Society Published on Web 07/13/2006
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Figure 1. Rheological characteristics of Porphyridium sp. polysaccharide (2%) b and hyaluronic acid (1%) O under conditions simulating walking and running, as affected by temperature.
Materials and Methods Alga Growth Conditions and Polysaccharide Production. Porphyridium sp. (UTEX 637) from the culture collection of University of Texas was cultivated in an artificial seawater (ASW)19 medium in polyethylene sleeves, as previously described.20 After 15-20 days, cultures were harvested by centrifugation (Cepa Z-41 20,000 rpm). The polysaccharide was then further treated by crossflow filtration to remove salts (e1 mS m-1) and to concentrate the polysaccharide (1%-2% w/v). Hyaluronic Acid. As a reference, we used hyaluronic acid (1% sodium hyaluronate), the active pharmaceutical ingredient (API) of the commercial product Arthrease (extracted from bacterial cells). The material was given by B.T.G ltd. Hyaluronidase Activity. Hyaluronidase activity was assayed viscometrically as previously described.21 Rheological Measurements. Rheological properties were determined in a Carri-Med CSL 50 controlled stress rheometer (TA Instruments, Leatherhead, U.K.) operated in cone-plate mode (cone angles 4°, diameter 40 mm). To evaluate the steady-shear viscosity as a function of shear rate, a given shear stress was initially applied, and the steady-state shear rate was then measured.22 A small amplitude oscillatory shear stress test was performed to measure the storage (elasticity) modulus G′ of the fluids.22 To assess viscoelasticity properties of the fluids under conditions relevant to knee movement, frequencies of 0.5 Hz (shear rate 3.14 s-1), associated with walking, and 2.5 Hz (15.7 s-1), associated with the rapid knee movements of running, were used.23 Differences between the viscosity (η) and elasticity (G′) properties were tested at different concentrations of Porphyridium sp. polysaccharide (1%, 2% w/v) and hyaluronic acid (0.5%, 0.75%, 1% w/v) at different temperatures (25, 37, 50, and 70 °C). Friction and Wear Measurements. Friction and wear of materials lubricated with polysaccharide solution (0.25% and 0.5%) or hyaluronic acid (1%) were compared in two types of experimental setups, i.e., ball-on-flat and ring-on-block. In the first set of experiments, a ceramic Si3N4 ball of diameter of 2 mm was allowed to slide against an alumina flat with reciprocal motion at a low sliding velocity of 0.2 mm s-1. This test was performed in order to (19) Jones, R. F.; Speer, H. L.; Kury, W. Physiol. Plant. 1963, 16, 636-643 (20) Cohen, E.; Arad (Malis), S. Biomass 1989, 18, 59-67. (21) Maksimenko, A. V.; Petrova, M. L.; Tischenko, E. G.; Schechilina, Y. V. Eur. J. Pharm. Biopharm. 2001, 51, 33-38. (22) Lapasin, R.; Pricl, S. Rheology of industrial polysaccharides; Blackie A & P: London, 1995. (23) Mensitieri, M.; Ambrosio, L.; Iannace, S.; Nicolais, L.; Perbellini, A. J. Mater. Med. 1995, 6, 130-137.
evaluate the behavior of the polysaccharide under severe contact conditions (virgin contact pressure, 0.5-1.5 GPa). The load was increased from 0.2 to 1.5 N, and the test was carried out over 500 cycles for each load. The change of friction force was measured as a function of time. In the second set of experiments, the contact conditions were chosen close to the knee joint: a flat UHMWPE block was allowed to slide against a hardened steel ring with a relatively high sliding velocity of 0.2 m s-1 over 2 h. Maximal virgin contact pressure was 5 MPa, and the temperature was held at 28 ( 1 °C. The rings were placed in a bath filled with the lubricant being tested. Atomic Force Microscopy (AFM). Freshly cleaved mica surfaces were exposed to aqueous solutions of polysaccharide and hyaluronic acid for 60 s, rinsed with copious amounts of water, and dried under a stream of pure nitrogen gas. Following adsorption, the surfaces were imaged under ambient conditions using a Digital Instruments (Veeco) Dimensions 3100 AFM operating in tapping mode.
Results and Discussion The purpose of this study was to evaluate Porphyridium sp. polysaccharidesin terms of its rheological and lubricant propertiessas a new stable biolubricant. A comparison of the elasticity and viscosity of aqueous solutions of Porphyridium sp. polysaccharide (1% and 2%) with hyaluronic acid (0.5 and 1%) showed that all samples tested displayed shear-thinning behavior (Figure 1). The solutions of Porphyridium sp. polysaccharide and hyaluronic acid exhibited different characteristic slopes for shear rate, temperatures and concentration (data not shown). The viscosity (η) and elasticity (G′) of the two biopolymers were compared at shear rates of 3.14 and 15.7 s-1 (frequencies of 0.5 and 2.5 Hz, respectively), i.e., values that correspond to running and walking movements of the knee.23 Porphyridium sp. polysaccharide (2%) and hyaluronic acid (1%) gave values of the same order of magnitude for viscosity and elasticity at room temperature (25 °C). When the temperature was increased, however, the viscosity and elasticity of hyaluronic acid decreased sharply, whereas the values for Porphyridium sp. polysaccharide decreased only moderately or even increased. For “walking” and “running” conditions, the elasticity of Porphyridium sp. polysaccharide decreased by 29% and 17%, respectively, when the temperature was increased from 25 to 70 °C, whereas for hyaluronic acid, reductions of 64% and 51%, respectively, were obtained. For “walking” and “running” conditions, the reduction
Superior Biolubricant from Red Microalga
Figure 2. Effect of the load on the friction coefficient in a ballon-flat device under severe contact conditions. The pairs were lubricated with hyaluronic acid (1%) -1, polysaccharide solutions (1%) - 2, and (2%) - 3. The test was carried out over 500 cycles. These experiments were carried out at a sliding velocity of 0.2 mm s-1.
in the viscosity of hyaluronic acid was 52% and 40%, respectively, when the temperature was increased from 25 to 70 °C, whereas for Porphyridium sp. polysaccharide a reduction in the viscosity of 14% was found for “walking” conditions and an increase of 12% for “running” conditions. Thus, unlike most lubricants, for which a rise in temperature is accompanied by a decrease in viscosity and hence a reduction of lubricity, the viscosity of the polysaccharide did not drop with rising temperatures (Figure 1). It is thus to be expected that tribological properties of the polysaccharide will be advantageous to those of hyaluronic acid. It seems that the macrostructure of the Porphyridium sp. polysaccharide becomes more highly organized with increasing temperatures, as opposed to hyaluronic acid and most other polysaccharides, which become less organized with increasing temperatures. This finding was not surprising, since the polysaccharide, unlike hyaluronic acid and chondroitin sulfates, does not contain the 1,4-linkages between 2-acetamido-2-deoxy-βD-glucose and D-glucose that are randomly hydrolyzed by hyaluronidase. The two biopolymers were also compared for their ability to reduce friction. In the ball-on-flat test, very low values of friction force were observed up to a load of 0.75 N for both hyaluronic acid and algal polysaccharide solutions. The appreciable advantage under friction of the polysaccharide was manifested at loads of 1-1.5 N: the friction force for the polysaccharide was significantly lower (approximately half) than that for hyaluronic acid at 1 N, and when the load was increased to 1.5 N, the friction force for hyaluronic acid became almost twice as highthan that for the polysaccharide. The dependence of the friction coefficient, µ, on the load for ball-on-flat pairs lubricated with the two compounds is shown in Figure 2. The values of µ for hyaluronic acid (1%) and polysaccharide (1% or 2%) were similar up to load of 0.7 N, being close to 0.003. It is therefore evident that the 1% polysaccharide solution showed the best friction properties under high loads. Both hyaluronic acid and the polysaccharide showed complicated dependences of the friction force F on the load P. These dependences did not follow Amonton’s law, F ) µP, where the friction coefficient remains constant with increasing load. Apparently, the contact conditions (contact area, structure of lubricant) change at the transition from low to high pressure. It is known that under high loads and low sliding velocities the main friction mechanism is boundary lubrication.24 It may be (24) Cameron, A. Principles of Lubrication; Longmans: London, 1966.
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Figure 3. Change in the friction force with time for ring-on-block pairs lubricated with 0.25% Porphyridium sp. polysaccharide solution (1) and 1% hyaluronic acid solution (2) Maximal virgin contact pressure was 5 MPa. These moderate contact conditions are similar to those in knee joints.
Figure 4. Optical micrographs of wear tracks on the surface of UHMWPE lubricated with hyaluronic acid (a) and polysaccharide solution (b). Ploughing tracks on the surface of the UHMWPE block indicate direct contact between the surfaces lubricated with hyaluronic acid.
expected that boundary lubrication is the dominant mechanism under low sliding velocity (0.2 mm s-1) and high pressure (>1 GPa with hyaluronic acid and polysaccharide lubricants). The low friction coefficient (∼0.003) is probably associated with the preservation of a thin layer of water at the interface under low loads. In this case, the friction behavior of hyaluronic acid is similar to that of the polysaccharide. As the load is increased, it is possible that water is squeezed out of the interface, with a concomitant decrease in the thickness of the lubricant layer. This decrease (in combination with the low adhesion of hyaluronic acid described below) increases the friction of the ceramic pair. The change in friction force with time for a hardened steel ring/ultrahigh-molecular-weight polyethylene (UHMWPE) block pair is shown in Figure 3. In this setup, the average values for the friction coefficients at the steady friction state were 0.025 and 0.05 for 0.25% polysaccharide and 1% hyaluronic acid solutions, respectively. The friction coefficients for the pair UHMWPE block-steel ring are seen to be significantly higher
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Figure 5. Atomic force microscopy (AFM) images of mica surfaces lubricated with polysaccharide solution, rinsed and dried. (a) 2% polysaccharide, 5-µm scan, z-scale 10 nm, (b) 0.1% polysaccharide, 5-µm scan, z-scale 10 nm; (c) 0.02% polysaccharide, 3-µm scan, z-scale 3 nm.
as compared to the ceramic pair. At the beginning of the test, the friction force with the polysaccharide was higher as compared to hyaluronic acid. It is expected that, under relatively low load (5 MPa), a thick film of the polysaccharide leads to high friction force. With time, the film is squeezed out and the thickness of the layer decreases, resulting in lower values of shear force. The wear rate for the pair lubricated with polysaccharide was significantly lower than that for hyaluronic acid, i.e., 3.2 mm3/ 105 cycles and 6.8 mm3/105 cycles for the polysaccharide and hyaluronic acid solutions, respectively. Optical microscopy showed that the wear track ploughed into the surface of the UHMWPE was much more pronounced for hyaluronic acid than for the polysaccharide (Figure 4). The lines observed at a higher magnification of the surface of the polysaccharide-lubricated UHMWPE block exposed to friction forces may be assumed to be polymer chains (Figure 4b). It is thus evident that the polysaccharide polymer strands had been preserved on the contact surfaces, whereas the hyaluronic acid had apparently been forced out of the space between the two surfaces. Wear tracks on the surface of UHMWPE block lubricated with hyaluronic acid confirmed this assumption. The above-described results are in keeping with previous studies showing that the lubrication performance of hyaluronic acid is impaired by poor adhesion to the shearing surfaces. Those studies were conducted on molecularly smooth mica surfaces in a surface forces apparatus (SFA). Hyaluronic acid failed to adhere to the shearing mica surfaces, which thus remained essentially unlubricated.2-4 To test whether an adhesion effect was responsible for the improved lubrication properties of the polysaccharide, experiments were carried out to compare the adsorption onto mica surfaces of the polysaccharide with that of hyaluronic acid. Freshly cleaved mica surfaces were exposed to aqueous solutions of polysaccharide or hyaluronic acid and then imaged by AFM under ambient conditions. The image of the mica surface exposed to a 2% polysaccharide solution showed the presence of a fairly compact layer of macromolecular strands (Figure 5a), with dark patches that represent voids in the layer. Section analysis of the void depths corresponded to a layer thickness of about 3 nm. For a 0.1% polysaccharide solution, a layer of polysaccharide strands was again observed on the surface, but the layer was less dense, and individual strands were clearly resolved (Figure 5b). Adsorption of a polysaccharide solution that had been even further diluted to 0.02% clearly showed single, isolated strands of the polymer adsorbed onto the surface (Figure 5c). Section analysis showed that the strand dimensions ranged from 0.5 to 0.8 nm in height and 22 to 30 nm in width. Experiments performed with aqueous solutions of hyaluronic acid under identical conditions showed no signs of the presence of an adsorbed layer on the mica surface (not shown). These findings provide support for the above-proposed premise that
Figure 6. Effect of hyaluronidase (0.2 mg/mL) on (a) a 0.02% solution of Porphyridium sp. polysaccharide and (b) 0.02% hyaluronic acid solution.
the inferior tribological properties of hyaluronic acid are a function of its lack of adhesivity.2-4 The stronger adsorption of the polysaccharide may be explained in terms of the charge of the mica and the polysaccharide. Freshly cleaved mica surfaces are negatively charged due to dissolution of potassium cations from the mica surface into the aqueous solution. Thus, the improved performance of the polysaccharide may be attributed to its stronger tendency to adsorb onto negatively charged surfaces, most probably due to suitable density of negatively charged groups in the polysaccharide that act as effective anchoring sites to the surface. Such behavior noticeably resembles the adsorption of DNA onto mica surfaces.25,26 Moreover, the presence of the noncovalently bound 66 kDa glycoprotein14 that is known to adsorb on mica is likely to enhance the binding of the polysaccharide onto the mica surface. It is thus likely that the friction damage to UHMWPE in the previous set of experiments was due to the poor adsorption of hyaluronic acid to the UHWMP surface. The presence of polysaccharide polymer strands adsorbed on the surfaces apparently contributed to the improved tribological properties obtained in the friction and wear tests. Experiments are currently under way to extend these adsorption studies to thermally treated sapphire (0001) surfaces.27 Since biolubricants are subjected to hyaluronidase activity in the human body, in vitro studies were used determine the ability of the polysaccharide to withstand degradation by this enzyme. (25) Bezanilla, M.; Manne, S.; Laney, D. E.; Lyubchenko, Y. L.; Hansma, H. G. Langmuir 1995, 11, 655-659. (26) Hansma, H. G.; Laney, D. E. Biophys. J. 1996, 70, 1933-1939. (27) Golan, Y.; Fini, P.; DenBaars, S. P.; Speck, J. S. Jpn. J. Appl. Phys. Part 1 1998, 37, 4695-4703.
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A change in viscosity was used as the measure of the resistance of the lubricant to hyaluronidase. Upon exposure of the two biolubricants to hyaluronidase activity, a 70% reduction in the viscosity of hyaluronic acid was determined after 10 min of incubation, whereas the viscosity of the Porphyridium sp. polysaccharide solution remained unchanged throughout the experiment (Figure 6). The advantages of the polysaccharide at high loads and high temperatures are in agreement with our previous studies showing the preservation of the double or triple helix polymer chains of polysaccharide that remain on the contact surfaces under friction.17,18 This suggestion was confirmed by AFM and optical microscopy studies, which showed that the polysaccharide is effectively adsorbed onto mica surfaces to form ultrathin
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(nanometer range) coating layers that can sustain lubrication of the contact area under extreme conditions of load and temperature. This action of the polysaccharide together with its resistance to degradation by hyaluronidase will contribute to its success as a lubricant that is superior to hyaluronic acid in various applications. Acknowledgment. The authors thank Prof. Jacob Israelachvili (UCSB) for enlightening discussions, Ms. I. Mureinik for her devoted editorial help and B.T.G. for donation of Arthrease samples. This work made use of the SPM facility of the R. Stadler Minerva Center for Mesoscale Macromolecular Engineering at BGU. LA060600X
