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Feature Article Inhibition of Protein Adsorption onto Silica by Polyvinylpyrrolidone S. Robinson and P. A. Williams* Centre for Water Soluble Polymers, The North East Wales Institute, Plas Coch, Mold Road, Wrexham LL11 2AW, U.K. Received April 18, 2002. In Final Form: August 7, 2002 Isotherms for the adsorption of poly(vinylpyrrolidone) (PVP) and bovine serum albumin (BSA) onto silica have been determined. The adsorption capacity for PVP increased with increasing molecular mass but was independent of pH and ionic strength. The adsorbed layer thickness, δ, was determined from ζ potential measurements. δ was found to increase with increasing surface coverage but was significantly less than the polymer radius of gyration even at the adsorption plateau, indicating that the polymer coils flattened at the surface. BSA did not adsorb onto silica from water at pH 7 because of electrostatic repulsions, but adsorption did occur in the presence of electrolyte. The amount of BSA adsorbed at plateau coverage increased from ∼0.1 mg/m2 in 0.001 M NaCl to ∼1.0 mg/m2 in 0.5 M NaCl. BSA adsorption was significantly inhibited but not completely prevented, by precoating the silica particles with PVP. Even at low PVP surface coverages, where δ had very low values, some inhibition occurred. Furthermore, PVP was able to desorb most but not all BSA molecules from the silica particles.
Introduction Poly(vinylpyrrolidone) (PVP) is an amphiphilic polymer and will readily dissolve in water and many nonaqueous solvents.1 It finds widespread application in a number of areas, for example, in aerosol products such as hair sprays, in glues, as a complexing agent for dyes to impart solubility, as a dispersing agent to provide colloid stability, and also as a blood plasma substitute.2 It has also been used in beer clarification for the removal of polyphenols.3 It has been shown that PVP is able to inhibit protein adsorption onto surfaces. For example, the coating of filtration membranes with PVP has been found to reduce fouling by bovine serum albumin (BSA)4,5 and lysozyme.6 The ability to inhibit protein adsorption is important in many other areas. For example, protein adsorption can result in the blocking of heat exchangers and in the buildup of hazardous bacterial films on working surfaces and equipment. In the medical field it can lead to the adhesion of platelets onto implants, resulting in thrombosis.7 As far as we are aware, there are no reports published which have investigated at the molecular level the role of PVP * To whom correspondence should be addressed. E-mail:
[email protected]. (1) Molyneux, P. Water soluble synthetic polymers: properties and behavior; CRC Press Inc.: Boca Raton, FL, 1983. (2) Budd, P. M. In Industrial Water Soluble Polymers; Finch, C. A., Ed.; RSC Special Publication no. 186; Royal Society of Chemistry: Letchworth, U.K., 1996; p 1. (3) The Brewers’ Handbook; Goldamer, T., Ed.; Apex publishers: Virginia USA, 2000. (4) Roesink, H. D. W.; Berrlage, M. A. M.; Potman, W.; Vandenboomgard, T.; Mulder, M. H. V.; Smolders, C. A. Colloids Surf. 1991, 55, 231. (5) Ko, M. K.; Pellegrino, J. J.; Nassimbene, R.; Marko, P. J. Membr. Sci. 1993, 76, 101. (6) Rovira-Bru, M.; Giralt, F.; Cohen, Y. J. Colloid Interface Sci. 2001, 235. (7) Chapman, R. G.; Ostuni, E.; Liang, M. N.; Meluleni, G.; Kim, E.; Yan, L.; Pier, G.; Warren, H. S.; Whitesides, G. M. Langmuir 2001, 17, 1225.
Table 1. Source of PVP Samples manufacturer
cat. no.
name
quoted mol. wt
ISP Technologies Inc Aldrich Chemicals Sigma Chemicals BASF
K-120 K80-100 K26-35 K-17
Plasdone K120 poly(vinylpyrrolidone) PVP-40T Kollidon 17PF
2 000 000 360 000 40 000 9 300
Table 2. Physicchemical Characteristics of the PVP Samples PVP sample
Mw
Mn
Mw/Mn
Rg
Rh
β
K-120 K80-100 K26-35 K-17
1 150 000 540 000 67 130 10 500
764 500 381 500 25 480 7 917
1.50 1.42 2.63 1.33
40.8 ( 1.5 32.6 ( 1.65
34.5 23.5 15.5 2.75
0.84 0.72
-
in inhibiting protein adsorption onto surfaces, and this, therefore, is the subject of this study. We have chosen silica as a model substrate. Surprisingly, relatively few papers appear in the literature concerned with the adsorption of PVP onto silica.8,9 Experimental Section Materials. PVP samples were obtained from a number of suppliers, and details are given in Table 1. Their molecular mass was determined by gel permeation chromatography (GPC) using a Superose 6 column with the Wyatt Technology Optilab Interferometric refractometer and Dawn DSP multiangle light scattering detectors. The solvent was 0.1 M NaNO3, and the flow rate was set at 0.5 mL/min. The Mw and Mn values and polydispersity index (Mw/Mn) are given in Table 2 together with the radius of gyration, Rg. The average hydrodynamic radius, Rh, of the PVP molecules was determined by photon correlation spectroscopy (PCS) using the Malvern Zetasizer 1000HAS. (8) Cohen Stuart, M. A.; Fleer, G. J.; Bijsterbosch, B. H. J. Colloid Interface Sci. 1982, 90, 310. (9) Thibault, A. M.; Misselyn-Baudin, A. M.; Broze, G.; Jerome, R. Langmuir 2000, 16, 9841.
10.1021/la020376l CCC: $22.00 © 2002 American Chemical Society Published on Web 10/10/2002
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Figure 1. ζ potential of silica particles as a function of pH in the presence of 1 × 10-3 M NaCl. Solutions of concentrations between 0.1 and 1.0 mg/mL PVP were used. Rh was plotted as a function of polymer concentration, and data were extrapolated to infinite dilution. The results obtained are given in Table 2. The values calculated for β (Rh/Rg) for K-120 and K80-100 are as expected for random coil polymers. Bovine serum albumin (BSA) was obtained from Sigma Chemicals. PCS measurements showed the BSA to be present as both monomers and dimers. The Rh of the monomer was found to be 1.8 nm, and that for the dimer was 4.7 nm. BSA has a molecular mass of 66 700, and the isoelectric point was determined from ζ potential measurements using a Malvern Zetamaster and found to be pH 4.4 in 1 × 10-3 M NaCl. Fumed silica was obtained from Wacker Chemicals Limited, Germany, and the surface area was quoted as 125 ( 15 m2 g-1. The ζ potential of the particles was determined using a Malvern Zetamaster in 1 × 10-3 M NaCl at 25 °C. The pH was adjusted using 1 × 10-3 M NaOH and HCl, and the results are plotted as a function of pH in Figure 1. Methods. Adsorption Isotherms. PVP Adsorption. Adsorption isotherms were produced using the depletion from solution method. PVP solutions (10 cm3) at varying concentration (0-2 mg/mL) were prepared and added to 0.1 g of silica. The dispersion was then sonicated for 1 min to ensure complete disaggregation of the silica particles and then tumbled for 24 h at room temperature. The samples were centrifuged at 2500 rcf using a MSE Mistral 3000i centrifuge, and the concentration of PVP in the resulting supernatant was determined by UV absorption at 210 nm. Further isotherms were produced at varying pH (310.5) and with varying electrolyte concentration (0.1-1.0 M NaCl). The PVP concentration for these isotherms was detected by complexation with iodine using the method of Levy and Fergus10 due to interference by the solvent at 210 nm. The preferential adsorption of varying molecular mass PVP species was determined by GPC using the system described above. Solutions (10 mL) of 1.0 mg/mL and 2.0 mg/mL PVP containing K-120 and K-17 in equal ratios were prepared and added to 0.1 g of silica and tumbled at room temperature (18 ( 2 °C) for 24 h. The molecular mass distributions of the original solution and the supernatant were determined. BSA Adsorption. The method used was similar to that described for PVP adsorption. Protein solution (10 cm3) in the concentration range 0-2 mg/mL was prepared, and 0.1 g of silica was added. The dispersions were sonicated for 1 min, tumbled for 24 h at room temperature, and centrifuged to separate silica from free protein. BSA has two UV absorption peaks, one at 210 nm and the other at 280 nm. The former coincides with the PVP UV absorption peak and hence, the amount of unadsorbed BSA present in the supernatant was therefore determined by direct UV absorption at a wavelength of 280 nm. Isotherms were produced in water alone and in the presence of NaCl in the concentration range 0-0.1 M. The solution pH was ∼7.0 for all samples. (10) Levy, G. B.; Fergus, D. Anal. Chem. 1953, 25, 1408.
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Figure 2. Adsorption isotherms for PVP samples of varying molecular weight at pH 7 onto silica from water: 1, K-17; 9, K26-35; b, K80-100; 2, K-120. Desorption of BSA on Silica by PVP. The silica particles were coated with BSA by adding 10 cm3 of BSA (0-2 mg/mL) to 0.1 g of silica in the presence of 0.01 M NaCl. The samples were washed three times by centrifuging, removing 8 cm3 of supernatant, adding 8 cm3 of 0.01 M NaCl, and redispersing. UV analysis of the first washing confirmed that BSA was not desorbed. 8 cm3 of PVP solution (0-2 mg/mL in 0.01 M NaCl) was then added to the BSA-coated silica particles dispersed in 0.01 M NaCl. The dispersions were tumbled for 24 h at room temperature and then centrifuged. The concentration of BSA desorbed was then determined by UV absorption. BSA Adsorption onto PVP-Coated Silica. Similar experiments were performed using PVP-coated silica particles adopting the same methodology. The concentration of BSA adsorbed onto the silica surface was determined using UV spectroscopy. Adsorbed Layer Thickness. ζ Potential Measurements. The ζ potential of the silica particles was determined as a function of the amount of PVP adsorbed using the Malvern Zetamaster. Experiments were conducted in 1 × 10-3 M NaCl at pH 4.5. The adsorbed layer thickness was determined using eq 1
( )
tanh
( )
zeζ1 zeψ0 -κ(δ-∆) ) tanh e 4kt 4kt
(1)
where z is the valence of the potential determining ions, e is the electron charge, ∆ is the thickness of the Stern layer (approximately 0.4 nm), and κ is the Debye-Huckel parameter, where 1/κ is the electrical double layer thickness with a value of κ ) 3.288xI for aqueous systems at 25 °C. The calculation assumes that the interfacial charge, surface potential, and charge distribution of ions within the diffuse layer are not significantly altered by the adsorbed polymer.
Results PVP Adsorption. The isotherms for the various PVP samples adsorbed onto silica at pH 7 are shown in Figure 2, and it is noted that the isotherms are high affinity and that the amount adsorbed increases with increasing molecular mass. The amount adsorbed at plateau coverage, Γ, is plotted as a function of log Mw in Figure 3, and data reported by Cohen Stuart et al.8 are included for comparison. The results show excellent agreement with Γ increasing sharply initially at molecular mass values < 105 but then reaching a pseudoplateau value. The isotherms of the K80-100 sample adsorbed onto silica in the presence of electrolyte and at varying pH are given in Figures 4 and 5, respectively, and show that the amount adsorbed is unaffected by changing the solvent conditions. The thickness of the adsorbed polymer layer, δ, determined from ζ potential measurements for K80-100 adsorbed onto silica, is plotted as a function of surface coverage in Figure 6. It can be seen that δ increases with
Inhibition of Protein Adsorption by Polyvinylpyrrolidone
Figure 3. Adsorption capacity for PVP on silica as a function of molecular mass: 9, from Cohen Staurt et al.;2 b, current data.
Figure 4. Adsorption isotherms for PVP (K80-100) onto silica at pH 7 from the following: 2, H2O; 9, 0.1 M NaCl; b, 0.5 M NaCl; 1, 1.0 M NaCl.
Figure 5. Adsorption isotherms for PVP (K80-100) onto silica at varying pH: b, pH 3.0; 9, pH 5.0; 2, pH 9.0; 1, pH 10.5.
increasing surface coverage, as predicted from theory,11,12 and attains a pseudoplateau value of ∼15 nm. This compares with an Rg of 32 nm for the molecules in solution and indicates that the molecular coils flatten (“pancake”) on adsorption on the surface. It is evident that at low coverage the molecules completely unfold at the surface and are present predominantly in trains. At higher coverages some unfolding still occurs but the polymer molecules adopt a more extended configuration with some loops and tails protruding out into solution. (11) Scheutjens, J. M. H. M.; Fleer, G. J. J. Phys. Chem. 1979, 83, 1619. (12) Scheutjens, J. M. H. M.; Fleer, G. J. J. Phys. Chem. 1980, 84, 178.
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Figure 6. Adsorbed layer thickness as a function of surface coverage for PVP (K80-100) adsorbed onto silica at pH 7.
Figure 7. Rg (2) and adsorbed layer thickness for PVP samples of varying molecular mass at surface coverages of 0.4 (1) and 1.0 (9).
Figure 8. Molecular mass distribution of PVP blend (K120/ K-17): 1, 0.1% PVP before adsorption; darker gray line, 0.1% PVP after adsorption; lighter gray line, 0.2% PVP after adsorption.
The values for δ at surface coverages of 0.4 and 1.0 as a function of PVP molecular mass are given in Figure 7 and are compared to values for Rg. The thickness of the adsorbed polymer layer increases with increasing molecular mass at both levels of coverage but is always less than Rg. The molecular mass distributions of the blend of K-120 and K-17 before and after adsorption onto silica are shown in Figure 8. It is noted that the high molecular mass species are preferentially adsorbed and that low molecular mass molecules are left unadsorbed in solution. BSA Adsorption. The adsorption of BSA onto silica is shown in Figure 9 as a function of electrolyte concentration.
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Figure 9. Adsorption isotherms for BSA onto silica with varying electrolyte concentration: [, no NaCl; b, 0.0001 M NaCl; 2, 0.001 M NaCl; 1, 0.01 M NaCl; 9, 0.15 M NaCl; ×, 0.5 M NaCl.
Figure 10. Adsorption isotherms for BSA onto PVP coated silica: 9, adsorption from H2O; 1, adsorption from 0.1 M NaCl.
There is no adsorption onto the silica particles from water or 0.0001 M NaCl. At higher electrolyte concentrations adsorption does occur and the isotherms show low affinity. The amount of BSA adsorbed at plateau coverage increases from ∼0.1 mg/m2 in 0.001 M NaCl to ∼1.0 mg/m2 in 0.5 M NaCl. Adsorption onto PVP- and BSA-Coated Particles. The isotherms for the adsorption of BSA onto silica particles coated with PVP (K80-100 sample) to plateau coverage from both water and 0.01 M NaCl are shown in Figure 10. It is noted that there is no or very little adsorption from water, as was the case for bare silica particles. In the presence of 0.01 M NaCl, BSA adsorption does occur but the amount adsorbed is significantly less than that for bare particles (0.25 mg/m2 compared to 0.8 mg/m2). When 10 cm3 of BSA solution at a fixed concentration (2.0 mg/mL) was added to silica particles (0.1 g) coated with varying amounts of PVP (K80-100) in the presence of 0.01 M NaCl, it was found that the amount of BSA adsorbed decreased dramatically with increasing PVP surface coverage (Figure 11). Both sets of experiments, therefore, suggest that BSA does not readily displace PVP from the surface. The addition of varying concentrations of PVP solution (K80-100) to silica particles fully coated with BSA from 0.01 M NaCl resulted in desorption of BSA from the silica surface (Figure 12). Maximum desorption occurred at PVP concentrations of 1.0 mg/mL and above with ∼75% of the BSA molecules being removed from the surface. Increasing
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Figure 11. Amount of BSA adsorbed onto silica particles coated with varying amounts of PVP from 0.01 M NaCl: O, adsorbed amount of BSA/mg‚m-2; 2, PVP adsorbed layer thickness/nm.
Figure 12. Desorption of BSA by PVP: 2, fraction of PVP adsorbed onto silica; b, fraction of BSA remaining adsorbed after addition of PVP.
the PVP concentration above 0.1% did not result in any further desorption. Discussion The isotherms for PVP adsorbing onto silica are high affinity, and the amount adsorbed at plateau coverage is consistent with data published in the literature.8,9 The adsorption capacity was found to be a function of molecular mass, showing a similar dependency to that reported by Cohen Stuart et al.8 GPC studies showed that high molecular mass material adsorbed preferentially over low molecular mass material. This is typically observed for the adsorption of nonionic polymers and is a consequence of the fact that the loss in configurational entropy is less for the higher molecular mass species. Calorimetric studies have indicated that the adsorption process is exothermic.9 FTIR studies on adsorption from deuterium oxide13 and dioxane14 have suggested that adsorption occurs through interaction of the PVP carbonyl groups and silanol groups on the silica surface. At low surface coverages the fraction of bound polymer segments has been reported to be ∼0.5, but this decreased to ∼0.25 at saturation coverage. Calorimetric studies have also indicated that the bound fraction is ∼0.25 at plateau coverage.9 Cohen Stuart et al.8 found that when the silica was subjected to heat treatment (900 °C), which removed a large proportion of the surface hydroxyl groups, the fraction of bound polymer (13) Day, J. C.; Robb, I. D. Polymer 1980, 21, 408. (14) Cohen Stuart, M. A.; Fleer, G. J.; Bijsterbosch, B. H. J. Colloid Interface Sci. 1982, 90, 321.
Inhibition of Protein Adsorption by Polyvinylpyrrolidone
segments decreased only slightly. When the silica surface hydroxyl groups were virtually all removed by reaction with an organosilicon, halide adsorption still occurred but the bound fraction was reduced to 0.02. They argued that this confirmed that the surface hydroxyls were involved in the adsorption process. In our studies we observed that the amount adsorbed was independent of the ionic strength and pH even though at pH 10 the surface sites will be largely in the form of silicate ions. Modeling of the conformation of the PVP chain has demonstrated that the amide carbonyl oxygens reside on one side of the polymer chain and are relatively exposed while the nitrogens are on the other side of the chain and are surrounded by methylene and methine groups.15 It was also noted that there is a significant positive dipole on the nitrogen and a negative dipole on the oxygen. Under conditions of high pH, therefore, it may also be that interaction occurs between surface silicate ions and the positive dipole on the PVP nitrogen atoms. Studies on the configuration of PVP molecules adsorbed onto silica using ESR16 and NMR17 have shown that the fraction of bound segments is >0.9 at low surface coverages and 0.5 at plateau coverage. These values are higher than those obtained by FTIR and calorimetry. This is expected, since both ESR and NMR distinguish between segments that are close to but not necessarily attached to the surface in trains and those extending away from the surface into solution in the form of loops or tails. FTIR and calorimetry only measure those segments in direct contact with the surface. The adsorbed layer thickness results obtained from the ζ potential measurements reported in this paper are consistent with the configurational data noted above, since at plateau coverage δ is considerably less than Rh of the PVP molecules, suggesting that mainly loops and trains are present.11,12 The amount adsorbed at plateau coverage and the adsorbed layer thickness both increase with increasing molecular mass of the PVP chains, as expected because of the development of longer loops and tails for the higher molecular mass molecules. BSA did not adsorb onto the silica particles from water at pH 7. At this pH the protein will have a net negative charge, and hence, it is likely that adsorption is prevented by electrostatic repulsions between the protein molecules and the silica surface. Similar conclusions were drawn by Shirahama and Suzawa for the adsorption of BSA onto acrylic acid/styrene latices.18 When the adsorption process was carried out in the presence of 0.001 M NaCl, some protein was found to adsorb (∼0.1 mg m-2). The electrolyte layer serves to reduce the thickness of the electrical double surrounding the particles and BSA molecules, enabling interaction to occur. Adsorption may take place through positive sites on the BSA molecules and silicate groups on the particle surface, or through hydrogen bonding and/or van der Waals attractive forces.19 BSA is easily denatured, and it has been reported that the driving force for the adsorption onto silica is the structural rearrangement of the molecules. It is envisaged that the polypeptide backbone of the BSA unfolds on the surface without exposing hydrophobic residues to water molecules. Structural rearrangement has been demonstrated by Giacomelli and Norde20 using circular dichroism and differential scanning calorimetry and by Su et al. using specular (15) Smith, J. N.; Meadows, J.; Williams, P. A. Langmuir 1996, 12, 3773. (16) Robb, I. D.; Smith, R. Polymer 1977, 18, 500. (17) Barnett, K. G.; Cosgrove, T.; Vincent, B.; Sissons, D. S.; Cohen Stuart, M. A. Macromolecules 1981, 14, 1018. (18) Shirahama, H.; Suzawa, T. Colloid Polym. Sci. 1985, 263, 141. (19) Haynes, C. A.; Norde, W. Colloids Surf. 1994, 2, 517.
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neutron reflection.21,22 BSA is ellipsoidal in shape with dimensions 11.9 nm × 2.7 nm × 2.7 nm.23 Su et al.21 found that the adsorbed layer thickness was always smaller than the dimensions of the short axis and suggested that the molecules adsorbed sideways on with their long axis parallel to the surface. For adsorption from 0.001 M NaCl it is calculated that the area occupied by the adsorbed BSA molecules is only ∼2.5% of the total area available. It is likely, therefore, that electrostatic repulsions between molecules in solution and the surface and lateral repulsions between the adsorbed BSA molecules restrict the amount adsorbed, as is seen generally for polyelectrolytes.24 Increasing the NaCl concentration to 0.01 M and above increased the amount adsorbed to ∼0.8 mg m2. This is expected, since the electrolyte will screen the repulsive forces. The results are in good agreement with the data obtained by others20,23,25 for BSA adsorption onto silica from 0.02 M phosphate buffer at pH 7. It is calculated that at plateau coverage BSA (using the nondenatured dimensions) would occupy ∼20% of the surface, thus supporting the concept that the BSA chains unfold. BSA is readily desorbed from silica by PVP, indicating that it has a much lower free energy of adsorption. PVP adsorption is an exothermic process due to bond formation through the carbonyl groups on the PVP and silanol groups on the silica. Microcalorimetric studies have reported that the fraction of bound segments is high (0.25)14 and hence the adsorption energy per molecule will also be high. The adsorption of BSA is likely to be an endothermic process19 and is governed by denaturation of the BSA chains. The adsorption energy per molecule is therefore expected to be much less. As indicated in Figure 10, the adsorption of BSA onto silica particles fully coated with PVP is significantly inhibited by the presence of adsorbed PVP molecules. It is evident, however, that some BSA molecules can penetrate the PVP layer and adsorb onto sites on the silica surface. McPherson et al.26 also found that the adsorption of fibrinogen and lysozyme onto silica was inhibited by grafting of poly(ethylene oxide) chains. However, they pointed out that some protein adsorption always occurred. Our results show that some inhibition occurs even at low PVP surface coverages (Figure 11), where the PVP molecules are essentially lying flat on the surface in trains. Steric repulsion between PVP loops and tails extending into solution and BSA molecules may help prevent protein adsorption but is clearly not a prerequisite. Interestingly, it has been shown that a high density of short chains of ethylene oxide (3-6 units) resists protein adsorption to an extent comparable to that for low densities of high molecular mass poly(ethylene oxide)s.26,27 McPherson et al.26 concluded that the mechanism for prevention of protein adsorption was the blocking of the protein adsorption sites. (20) Giacomelli, C. E.; Norde, W. J. Colloid Interface Sci. 2001, 233, 234. (21) Su, T. J.; Lu, J. R.; Thonas, R. K.; Cui, Z. F.; Penfold, J. J. Phys. Chem. B 1998, 102, 8100. (22) Su, T. J.; Lu, J. R.; Thomas, R. K.; Cui, Z. F. J. Phys. Chem. B 1999, 101, 3727. (23) Norde, W.; Anusiem, A. C. I. Colloids Surf. 1992, 66, 73. (24) Harrison, I. M.; Meadows, J.; Robb, I. D.; Williams, P. A. J. Chem. Soc., Faraday Trans. 1995, 91, 3919. (25) Norde, W.; Favier, J. P. Colloids Surf. 1992, 64, 87. (26) Mcpherson, T.; Kidane, A.; Szleifer, I.; Park, K. Langmuir 1998, 14, 176. (27) Chapman, R. G.; Ostuni, E.; Yan, L.; Whitesides, G. M. Langmuir 2000, 16, 6927.
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Conclusions PVP adsorbs onto silica particles through interaction of the carbonyl groups of the pyrrolidone rings and silanol groups on the silica surface. The adsorption capacity is dependent on the PVP molecular mass but is independent of ionic strength and pH. The adsorbed layer thickness is less than the polymer Rg, indicating that the molecular coils flatten at the surface. BSA does not adsorb onto silica from water because of charge repulsions, but adsorption
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does occur in the presence of electrolyte. PVP inhibits the adsorption of BSA onto silica but does not completely prevent it. PVP molecules can readily displace ∼75% of the BSA adsorbed on the silica. Acknowledgment. The authors are grateful to EPSRC and Reckitt Benckiser for support for this project. LA020376L
