J. Phys. Chem. 1996, 100, 16771-16775
16771
Biomolecular Recognition at Phospholipid-Covered Polystyrene Microspheres Sandra Miceli Sicchierolli and Ana Maria Carmona-Ribeiro* Departamento de Bioquı´mica, Instituto de Quı´mica, UniVersidade de Sa˜ o Paulo, CP 26077, Sa˜ o Paulo SP, Brazil ReceiVed: June 17, 1996; In Final Form: August 8, 1996X
Cholera toxin and its receptor (the monosialoganglioside GM1) are used as a model pair to evaluate phospholipid-covered polystyrene microspheres regarding their suitability as a matrix to promote biomolecular recognition. Toxin binding to GM1/phosphatidylcholine-covered polystyrene microspheres occurs in a molar proportion of 1:1 (toxin/GM1) and is strictly dependent on GM1 incorporation in the phospholipid-covered latex. Phospholipid coverage on latex offers a proper environment to GM1/toxin recognition. Phospholipid assembly on the latex surface depends on the physical state of the bilayer. A dipalmitoylphosphatidylcholine (DPPC) monolayer coverage on latex at room temperature can be obtained by incubating latex and DPPC vesicles in buffer solution for 1 h at 65 °C. Nonspecific physical adsorption of cholera antitoxin on latex surfaces can be controlled by covering latex with phospholipids. Upon increasing dipalmitoylphosphatidylcholine (DPPC) concentration in latex dispersions, nonspecific cholera antitoxin adsorption on latex decreases. This provides a way of modulating protein adsorption on the DPPC-covered latex. Changing the hydrophobichydrophilic character of the antitoxin itself by covalent attachment of an N-acyl residue increases physical adsorption on bare latex and decreases it on phospholipid-covered latex. The results may be of importance in development of immunoassays and biosensors for amplification of biomolecular recognition.
Introduction Exogenous particles such as liposomes, emulsion droplets, nanoparticles and other colloidal drug carriers have their clearance initialized in ViVo by adsorbing serum proteins, the so-called opsonins. Serum proteins include albumin, immunoglobulins, complement components, and adhesins such as fibrinogen and fibronectin. An inverse correlation has been found between the amount of proteins adsorbed from serum and the circulation half-life of colloidal drug carriers.1-3 Despite the importance of opsonization, solid-phase immunoassays, immobilization of enzymes, building-up of biosensors, and protein interactions with membranes and solid surfaces, in general, surprisingly little is known of the physicochemical factors determining modulation of protein adsorption on a variety of native and modified surfaces.4-7 The hydrophobic, van der Waals, electrostatic, and hydrogen-bonding interactions are the most important interaction types involved in physical adsorption of proteins on surfaces. Proteins or surfaces may also be chemically modified so that the effects of the chemical modification on the adsorbed amount can be determined.8 A less explored approach regards the effects phospholipids may have in modulating protein adsorption on model surfaces and how this could be useful in connection to amplification of biomolecular recognition by polystyrene microspheres. The interaction between phospholipids and polystyrene microspheres was recently described.9 Amidine polystyrene microspheres are first covered with a phospholipid monolayer due to the dominance of the hydrophobic interaction between latex and the phospholipid bilayer. Thereafter, phospholipid bilayers deposit onto the monolayer-covered latex due to the van der Waals attraction.9 We took advantage of the phospholipid coverage to promote incorporation of the cholera toxin receptor, i.e., the monosialoganglioside GM1, onto polystyrene microspheres.10 Quantitative analysis of the total incorporation carried out using pyrene-labeled GM1 yielded 50% incorpora* To whom correspondence should be addressed. X Abstract published in AdVance ACS Abstracts, September 15, 1996.
S0022-3654(96)01785-6 CCC: $12.00
TABLE 1: Properties of the Sulfate (SULF) and Amidine (AM) Polystyrene Microspheres in Watera latex
mean diameter/nm
area per charge group/nm2
SSA/(cm2 g-1)
sulf AM AM
249 193 172
772 374 473
228 401 294 673 330 651
a
SSA are the specific surface areas.
tion of the total GM1 amount added when microspheres were covered with one monolayer plus one bilayer of phosphatidylcholine.10 In contrast, GM1 did not adsorb on the bare latex as expected for the interaction between the hydrophilic sialosyloligosaccharide moiety of GM1 and the hydrophobic latex surface.10 In this work, we demonstrate that incorporation of GM1 in phospholipid-covered latex preserves its ability to specifically bind cholera toxin. Phospholipid coverage on latex and protein acylation are shown to modulate protein adsorption by decreasing or increasing, respectively, adsorbed protein amounts on bare latex. Material and Methods Egg phosphatidylcholine (PC) (type XIII-E) and dipalmitoylphosphatidylcholine (DPPC) were obtained from Sigma. PC or DPPC concentrations were determined by inorganic phosphorus analysis.11 Amidine or sulfate microspheres described as ultraclean by the supplier were obtained from the Interfacial Dynamics Corp. and were used as supplied. Properties of the microspheres are in Table 1. Water was of MILLI-Q quality. Vesicles and particles were buffered at pH 7.4 in 10 mM Tris(hydroxymethyl)aminomethane (TRIS) solutions. TRIS was obtained from Sigma. Monosialoganglioside (GM1) available as a lyophilized powder approximately 95% pure was obtained from bovine brain (Sigma). Cholera toxin (87 kDa) from Vibrio cholerae available as a lyophilized powder and delipidized anticholera toxin as whole antiserum developed in rabbit were obtained from Sigma. © 1996 American Chemical Society
16772 J. Phys. Chem., Vol. 100, No. 41, 1996 Small unilamellar PC or DPPC vesicles with a mean diameter of 60 and 74 nm, respectively,9 were prepared in 10 mM TRIS, pH 7.4, by ethanol injection12,13 followed by 4 h dialysis against 0.5 L of the same buffer in order to eliminate ethanol from the dispersion. The interaction between vesicles and microspheres was induced by adding vesicles to the latex. For PC, mixtures were thermostated at 25 °C for 24 h.10 For DPPC, they interacted at 25 or 65 °C for 1 h. Thereafter, they were centrifuged at 20000g for 1 h at 15 °C to separate the particles from the vesicles. The supernatant was used to determine phospholipid concentration. The adsorption isotherm for DPPC onto the microspheres was obtained from inorganic phosphorus analysis in the supernatant and in the vesicle dispersion. The total surface area on the polystyrene was calculated from the particle number density and the mean particle radius from electron microscopy, as given by the supplier. Adsorption was expressed as the number of phospholipid molecules adsorbed per square meter of polystyrene. The limiting adsorption and area per adsorbed molecule at limiting adsorption were determined from the plateau level on the isotherm. The interaction between GM1 and phospholipid-covered microspheres was induced by adding a 0.01 mg/mL GM1 solution in 10 mM TRIS, pH 7.4, to the covered latex. After 0.5 h interaction time to allow spontaneous insertion of the GM1 molecule on the phospholipidic microsphere coverage,10 the mixture was used to evaluate toxin binding to the phospholipid/ GM1-covered microspheres. The interaction between antitoxin and bare latex in TRIS 10 mM, pH 7.4, was induced by adding a solution of antitoxin to the microsphere dispersion. Mixtures were thermostatted at 37 °C for 1 h before centrifugation (14000g, 15 °C, 1 h) to separate particles from antitoxin in the supernatant. Protein in the supernatant was analytically determined using Lowry’s method.14 Protein adsorption was expressed in milligrams adsorbed protein per square meter of polystyrene. A similar procedure was followed for DPPC-covered latex. Latex was covered with DPPC by incubating vesicles and latex at 65 °C for 1 h. Derivatized cholera antitoxin was obtained from N-hydroxysuccinimide ester of palmitic acid following a procedure previously described to incorporate the protein in liposomes (page 84, ref 15). Fatty acid chains were attached via amide linkages to various sites on the protein so that the hydrophobicity of the antibody molecule was increased. Typically, 20 mg of sodium deoxycholate in 0.5 ml of 10 mM phosphate buffered saline was transferred to 20 µg of palmitic acid ester, added to 0.5 mL of a 5 mg/mL antitoxin solution, and incubated overnight at 37 °C in a rotating mixer. Thereafer, the N-acyl antitoxin was ready for use. N-acyl protein and bare or phospholipidcovered latex were mixed and allowed to interact following the procedure described above for the nonderivatized protein. Results and Discussion 1. Specific Binding of Cholera Toxin to GM1/Phospholipid-Covered Latex. Figure 1 shows binding of cholera toxin to GM1/PC-covered polystyrene microspheres as a function of total toxin amount added to the mixtures. In the absence of GM1, the toxin, a water-soluble protein of 84 000 kDa,16 does not bind to the PC-covered microspheres (Figure 1). Therefore, cholera toxin binding obtained in Figure 1 is specifically due to the biomolecular recognition between the toxin and its receptor. Further confirmation of specific binding can be achieved from a direct comparison between total numbers of (1) GM1 molecules on the covered latex; (2) toxin molecules at limiting toxin binding. The last amount (Figure 1B) is about
Sicchierolli and Carmona-Ribeiro
Figure 1. Cholera toxin binding to phosphatidylcholine (PC) covered latex in the presence (b) or in the absence (4) of its specific receptor, the monosialoganglioside GM1. There is no binding in the absence of GM1 (A). Final PC concentration is 0.1 mM for mixtures containing 1010 amidine polystyrene microspheres (172 nm mean diameter). Micrograms of bound protein in A, percent of bound protein in B (9), or milligrams of bound protein per square meter of polystyrene in C (9) represent toxin binding as a function of the total amount of toxin added to the mixtures.
60% of the total toxin amount in the mixture, i.e., 60% of 35 µg. Thus, 21 µg of cholera toxin, i.e. 14.4 × 1013 toxin molecules, is bound to GM1 incorporated on the phosphatidylcholine-covered microspheres. The total amount of GM1 in the mixture is 39.1 × 1013 GM1 molecules (2.6 µM GM1 in 0.25 mL). The area per GM1 molecule in a GM1 monolayer at the air/water interface is 0.6 nm2,17 whereas the area per GM1 molecule at maximal incorporation on a PC-covered polystyrene microsphere is 5.7 nm2.10 The total GM1 amount on phospholipid-covered latex can now be calculated. The total surface area on 1010 AM172 microspheres covered with a PC monolayer plus a DPPC bilayer is 0.001 15 m2, if the total surface area on the particles takes into account a particle radius of 86 + 2.5 + 2.0 + 5.0 nm (microsphere radius + monolayer thickness + equilibrium separation distance between monolayer and bilayer + bilayer thickness).10 Taking 5.7 nm2 as the area per GM1 molecule on covered latex, 0.00115/(5.7 × 10-18), there will
Phospholipid-Covered Polystyrene Microspheres
Figure 2. Antitoxin adsorption on bare polystyrene microspheres for two different latex types. For equivalent total surface areas on amidine (AM 172 nm) and sulfate polystyrene microspheres (SULF 249 nm), antitoxin adsorption is higher for the latter microsphere type. Upon an increase in particle number density, the amount adsorbed per square meter of amidine polystyrene substantially decreases.
be 2 × 1014 GM1 molecules inserted in the outer PC bilayer on latex at maximal incorporation. This corresponds to 51.1% of the total amount of GM1 in the mixture, in very good agreement with the 55% maximal incorporation reported previously.10 For a GM1/toxin proportion of 1:1, 2 × 1014 toxin molecules should be bound to GM1 on the microspheres. At limiting toxin binding, 1.5 × 1014 toxin molecules are bound (Figure 1), in fair agreement with the calculated amount of 2 × 1014 GM1 molecules on the PC-covered latex. 2. Nonspecific Adsorption of Cholera Antitoxin to Bare or Phospholipid-Covered Polystyrene Microspheres. Figure 2 shows adsorption isotherms in 10 mM TRIS, at pH 7.4, for delipidized whole cholera antiserum which contains the cholera antitoxin on positively and negatively charged polystyrene microspheres, AM172 (4 × 1010 parts/mL) and SULF249 (6.3 × 1010 parts/mL), respectively, at equivalent total areas on the latex surface. Separation of serum proteins by cellulose acetate electrophoresis at pH 8.6 is of current clinical use to identify abnormal immunoglobulins often occurring in certain diseases. At pH 8.6, serum normality is identified by all serum proteins being negatively charged (including the immunoglobulins) and migrating to the positive pole. The immunoglobulins are the less negatively charged serum proteins at pH 8.6. At pH 7.4, this negative charge is expected to be even lower. Under our experimental conditions (10 mM TRIS, pH 7.4), which are not suitable to perform protein electrophoresis, it is difficult to ascertain if the immunoglobulins are positive, neutral, or negatively charged. For the same total surface area on latex, cholera antitoxin adsorption on positively charged latex is greater than its adsorption on the oppositely charged sulfate microspheres (Figure 2). This would be consistent with serum proteins being negatively charged and the electrostatic attraction making a positive contribution to protein adsorption on latex. Nevertheless, the importance of the hydrophobic attraction in determining adsorption of the negatively charged serum proteins on the sulfate polystyrene cannot be overlooked, since it is still determining large adsorbed amounts of antitoxin on the negatively charged latex (Figure 2). Increasing particle number density, the total surface area available for adsorption on amidine
J. Phys. Chem., Vol. 100, No. 41, 1996 16773
Figure 3. Effect of the bilayer physical state on adsorption of dipalmitoylphosphatidylcholine (DPPC) from small vesicles on amidine polystyrene microspheres (AM172) in 10 mM TRIS, pH 7.4. Interaction between vesicles and microspheres was carried out for 1 h at 25 (9) or 65 °C (b). At these temperatures the DPPC bilayer is in the gel and in the liquid-crystalline state, respectively. Thereafter, centrifugation separated particles from vesicles, and DPPC was analytically determined in the supernatant. Final particle number density in the mixtures is 4 × 1011 particles/mL.
latex increases and the total protein amount adsorbed per square meter of polystyrene can be substantially decreased (Figure 2). Because the hydrophobic attraction between latex and protein seems to be the most important driving force for protein adsorption, one should expect conformational changes in the adsorbed protein that would eventually lead to formation of a thick and possibly disordered protein film on latex. Certainly, this could be detected as a loss of protein ability for antigen recognition upon its adsorption on latex. In fact, this has been one of the major drawbacks hampering latex use for the amplification of biomolecular recognition.18 Our rationale for designing the experiments presented next was that latex hydrophobicity had to be circumvented to decrease conformational changes in the protein molecule caused by the hydrophobic adsorption. In Figure 4, we took advantage of dipalmitoylphosphatidylcholine adsorption on latex as a monolayer (Figure 3) to modulate antitoxin adsorption. There is a decrease of antitoxin adsorption on latex as the total DPPC concentration in the mixtures is increased (Figure 4). It is important to emphasize that the DPPC adsorption constant for amidine latex is ca. 10 times larger than that for PC.9 At least 24 h interaction between particles and vesicles is required for latex coverage with PC.9,10 To reduce the interaction time required for coverage, instead of using PC, the use of DPPC was attempted due to its higher affinity for the latex. At room temperature, adsorption isotherms for DPPC (1 h interaction time) did not indicate any specific type of DPPC assembly on latex (Figure 3). The high adsorption constant leads to large amounts adsorbed possibly due to entire vesicle adhesion on latex. No plateau levels indicative of monolayer, trilayer or pentalayer coverage10 are present in the isotherm obtained at room temperature (Figure 3). On the other hand, if the latex/ vesicle interaction is carried out at 65 °C, i.e., above the phase transition temperature of the DPPC bilayer, a convenient DPPC monolayer coverage at limiting adsorption is obtained (Figure 3). The plateau level corresponds to 20 × 1017 molecules
16774 J. Phys. Chem., Vol. 100, No. 41, 1996
Figure 4. Modulation of cholera antitoxin adsorption on latex AM172 by dipalmitoylphosphatidylcholine (DPPC). Antitoxin adsorbed per square meter of polystyrene is shown as a function of antitoxin amount in the supernatant at different DPPC concentrations in TRIS buffer, pH 7.4. Final particle number density is 4 × 1011 parts/mL.
adsorbed per square meter polystyrene. This figure is consistent with an area per DPPC molecule on latex equal to 0.5 nm2, which is precisely the limiting area per monomer in a DPPC monolayer at the air/water interface. At limiting adsorption, there is one DPPC monolayer adsorbed on amidine latex. Thereafter, interaction at 65 °C for 1 h between vesicles and particles was used as the routine protocol to cover amidine latex with DPPC. The effect of changing the physical state of the DPPC bilayer to the more fluid liquid-crystalline state is to speed up reorganization of the DPPC molecules on the latex surface so that the bilayer structure in the vesicle assembly turns into a monolayer coverage. Figure 3 shows that it is difficult to achieve molecular reorganization on the surface when interaction is carried out with the DPPC bilayer in the rigid gel state (25 °C). Entire vesicle adhesion without occurrence of vesicle rupture was previously described for vesicles in the rigid gel state that interacted with oppositely charged polymeric19 or biological surfaces.20 DPPC coverage on latex promotes a decrease in antitoxin adsorption (Figure 4). We are currently investigating the preservation of antitoxin functionality as a function of DPPC concentration in the latex/phospholipid/antitoxin mixture. 3. Modulation of N-Acyl Cholera Antitoxin Adsorption on Latex by Phospholipid Coverage. The effect of negative modulation by DPPC was significantly amplified by increasing the hydrophobic character of the protein itself. Figure 5 shows adsorption isotherms for the N-acyl derivative of cholera antitoxin at three different DPPC concentrations: zero, very small, and 0.59 mM DPPC. The range of modulation is wider than that obtained for the nonderivatized protein as depicted from the comparison between Figures 4 and 5. The amount of N-acyl protein adsorbed is very sensitive to the presence of DPPC. In Figure 6, a final DPPC concentration as small as 0.03 mM decreases N-acyl antitoxin adsorption by 30%. Such a small final concentration of DPPC certainly is not enough to cover all polystyrene microspheres present in the dispersion with one monolayer. In accordance with the adsorption isotherm indicative of monolayer coverage (Figure 3), at 4 × 1011 parts/ mL, the final DPPC concentration in the mixture required to obtain monolayer coverage would be 0.18 mM or higher. This decreases adsorption by about 80% of the amount on bare latex
Sicchierolli and Carmona-Ribeiro
Figure 5. Modulation of N-acyl cholera antitoxin adsorption on latex AM172 by dipalmitoylphosphatidylcholine (DPPC). N-acyl cholera antitoxin adsorbed per square meter of polystyrene is shown as a function of protein amount in the supernatant. Latex and DPPC were mixed to a final concentration of 0.59 mM DPPC and a final particle number density of 4 × 1011 parts/mL (1 h, 65 °C). Thereafter, the mixture was centrifuged, and the supernatant was removed. Resuspension was performed in protein solutions that did not contain DPPC. A second centrifugation allowed the determination of protein remaining in the supernatant (b). Protein interacting with covered latex without supernatant removal yielded a much smaller adsorption amount (9). As a control, protein adsorption on bare latex is shown (2).
Figure 6. Modulation of N-acyl cholera antitoxin adsorption on latex AM193 by dipalmitoylphosphatidylcholine (DPPC). Final DPPC concentration is 0 (9) and 0.03 mM (b). Protein acylation and phospholipid coverage modulate physical antitoxin adsorption on latex.
(Figure 5). If there is a proportionality between decrease in adsorption and total amount of latex covered with DPPC, at 0.03 mM DPPC, adsorption should be reduced by about 13% relative to the amount on bare latex. In Figure 6, adsorption decrease is about 30%, i.e. much higher than expected. It is possible that covalent attachment of the N-acyl residue to the protein has changed not only its affinity for hydrophobic surfaces but also its solubility in water solution. Formation of hydrophilic N-acyl protein clusters could explain the change in affinity for the covered latex surface, which should still be hydrophobic by adding only 0.03 mM DPPC. Changing the hydrophobichydrophilic character of solid surfaces seems to be of more predictable consequences than changing it for more flexible and changeable biopolymer structures as those belonging to proteins. Presently, we are investigating the effects phospholipid adsorp-
Phospholipid-Covered Polystyrene Microspheres tion and protein derivatization may have on biomolecular recognition between cholera toxin (bound to GM1/DPPC on latex) and its antitoxin (bound to bare or DPPC-covered latex). Conclusions Biomolecular recognition between cholera toxin and its receptor GM1 is preserved by incorporating GM1 in phospholipid-covered polystyrene microspheres. Nonspecific physical adsorption of cholera antitoxin on latex surfaces can be controlled by covering latex with phospholipids. Phospholipids decrease nonspecific antitoxin adsorption on latex. Changing the hydrophobic-hydrophilic character of the antitoxin by acylation increases protein adsorption on bare latex and decreases protein adsorption on phospholipid-covered latex. Acknowledgment. Research grants 93-2288-6 and 960704-0 from FAPESP; 510022/93-6 and 520130/94-4 from CNPq are gratefully acknowledged. S. M. S. is the recipient of a FAPESP undergraduate fellowship (94/3721-8). References and Notes (1) Chonn, A.; Semple, S. C.; Cullis, P. R. J. Biol. Chem. 1992, 267, 18759. (2) Gregoriadis, G. in Stealth Liposomes; Lasic, D., Martin, F., Eds.; CRC Press: Boca Raton, FL, 1995; pp 7-12. (3) Storm, G.; Belliot, S. O.; Daemen, T.; Lasic, D. D. AdV. Drug DeliVery ReV. 1995, 17, 31.
J. Phys. Chem., Vol. 100, No. 41, 1996 16775 (4) Norde, W. AdV. Colloid Interface Sci. 1986, 25, 267. (5) Haynes, C. A.; Norde, W. Colloids Surf. B. 1994, 2, 517. (6) Malmsten, M.; Van Alstine, J. M. J. Colloid Interface Sci. 1996, 177, 502. (7) Malmsten, M. J. Colloid Interface Sci. 1995, 172, 106. (8) Malmsten, M.; Lassen, B.; Van Alstine, J. M.; Nilsson, U. R. J. Colloid Interface Sci. 1996, 178, 123. (9) Carmona-Ribeiro, A. M.; Herrington, T. M. J. Colloid Interface Sci. 1993, 156, 19. (10) Sicchierolli, S. M.; Carmona-Ribeiro, A. M. Colloids Surf. B. 1995, 5, 57. (11) Houser, G.; Fleischer, S.; Yamamoto, A. Lipids 1970, 5, 494. (12) Batzri, S.; Korn, E. D. Biochim. Biophys. Acta 1973, 298, 1015. (13) Kremer, J. N. H., Esker, M. W., Pathmamanoharan, C.; Wiersema, P. H. Biochemistry 1977, 16, 3932. (14) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193, 265. (15) New, R. R. C. In Liposomes, A Practical Approach; New, R. R. C., Ed.; IRL Press: Oxford, U.K., 1990, p 84. (16) Lospalluto, J. J.; Finkelstein, R. A. Biochim. Biophys. Acta 1972, 257, 158. (17) Luckham, P.; Wood, J.; Froggat, S.; Swart, R. J. Colloid Interface Sci. 1993, 156, 164. (18) Hermanson, G. T.; Mallia, A. K.; Smith, P. K. Immobilized Affinity Ligand Techniques; Academic Press: San Diego, 1992. (19) Tsuruta, L. R.; Lessa, M. M.; Carmona-Ribeiro, A. M. Langmuir 1995, 11, 2938. (20) Tapias, G. N.; Sicchierolli, S. M., Mamizuka, E. M.; CarmonaRibeiro, A. M. Langmuir 1994, 11, 3461.
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