Langmuir 2000, 16, 1457-1459
Reversible Cell Aggregation Induced by Specific Ligand-Receptor Coupling Wolfgang Meier Institut fu¨ r Physikalische Chemie, Departement Chemie, Universita¨ t Basel, Klingelbergstrasse 80, CH-4056 Basel, Switzerland Received July 12, 1999. In Final Form: September 27, 1999
Introduction It is well-known that the hydrophobic anchor groups of so-called hydrophobically modified water-soluble polymers, i.e., polymers that carry a low fraction of hydrophobic groups along an otherwise water-soluble polymer backbone, can be inserted into the lipid membranes of vesicles or even biological cells.1 As a result, the polymers are immobilized at the surface of the lipid membrane. The driving force for this process is the energy gain associated with transferring the hydrophobic groups of the polymer from a polar, at least partially water-exposed environment in the aqueous solution, into the hydrophobic part of a lipid membrane. This can, for example, be used to sterically stabilize lipid vesicles2 or, as recently shown, to interconnect different lipid membranes by a bridging polymer.3,4 For example, the two hydrophobic end groups of R,ωhydrophobically modified poly(ethylene oxides) carrying cholesteryl end groups can be inserted either into the bilayer membrane of the same particle, thus forming a loop, or into the membranes of two different particles, thus leading to a “bridge”. Usually the formation of loops is entropically favored compared to the bridges.4 Nevertheless, if the interparticle distances in the system are comparable to the dimensions of the polymer, a considerable fraction of the molecules is available for bridging. This can be exploited to prepare physically cross-linked hydrogels from lipid vesicles with an interesting polymervesicle hybrid network structure.3 Similarly, also biological cells can be interconnected by such polymers, thus leading to a polymer-induced cell agglomeration already at rather low polymer concentrations.3 Hence, these polymers can be regarded as a primitive model for the so-called cell adhesion molecules (CAMs)5,6 and have indeed recently found interest in the area of tissue engineering.7 CAMs are usually transmembrane proteins which are able to dock via a specific molecular recognition process (mostly a ligand-receptor bond) to proteins at the surface of other cells or to the extracellular matrix. Hence, they play an important role in cell recognition of the immune system or the formation of tissue.5,6 However, compared to the CAMs the R,ω-hydrophobically modified PEOs are neither effective (e.g., due to loop formation) nor specific for cell agglomeration. It is straightforward that this could be improved using a system more closely related to the natural model, e.g., unsym(1) Ringsdorf, H.; Schlarb, B.; Venzmer, J. Angew. Chem. 1988, 100, 117. (2) Lasic, D. D. Liposomes: From Physics to Applications; Elsevier Science Publications B.V.: Amsterdam, The Netherlands, 1993. (3) Meier, W.; Hotz, J.; Gu¨nther-Ausborn, S. Langmuir 1996, 12, 5028. (4) Lipowsky, R. Colloid Surf., A 1997, 128, 255. (5) Bell, G. I.; Dembo, M.; Bongrand, P. Biophys. J. 1984, 45, 1051. (6) Dembo, M.; Bell, G. I. Curr. Top. Membr. Transp. 1987, 29, 71. (7) Edelman, G. M. Science 1983, 219, 450.
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metrically substituted PEOs carrying only at one end a hydrophobic anchor group and at the other one a specific ligand or receptor group available for molecular recognition. Recently a similar reversible aggregation of vesicles induced by means of site specific ligand-receptor coupling has been described,8-10 e.g., a metal-ligand recognition process9 or the coupling between biotinylated lipids and streptavidin.10 Streptavidin is known to possess four binding sites for biotin and consequently is able to act as a linker between biotinylated lipids within different vesicles. In contrast to lipid vesicles, the surface of biological cells is, however, additionally covered by a polysaccharide layer of about 10-20 nm thickness, i.e., the glycocalix.5 Hence, for example, the biotin group of a conventional biotinylated lipid is expected to be hidden below this surface layer and consequently not accessible for the interaction with streptavidin due to a steric repulsion between the bulky protein and the polysaccharide layer. It is straightforward that one has to introduce a watersoluble spacer between the hydrophobic anchor group and the water-soluble biotin group, which is long enough to overcome the glycocalix. In this paper we report the synthesis of a series of unsymmetrically substituted PEOs carrying at one end a hydrophobic cholesteryl group and at the other end a hydrophilic biotin group (Chol-PEO-Bio). These polymers can be anchored via their hydrophobic cholesteryl group in the lipid membrane of biological cells. Depending on the length of the PEO spacer, the cells can be reversibly aggregated using the interaction between streptavidin and the biotinylated end of the polymer. Experimental Section Preparation of the Unsymmetrically Substituted PEOs (Representative Example). Cholesterol (0.25 mmol, 97 mg) was dissolved in 30 mL of dry tetrahydrofuran (THF) and a THF solution of K-Naphthalide was added under an argon atmosphere until the solution adopted a slightly green color which was stable for a period of more than 10 min. Afterward 200 mmol (10 mL) of ethylene oxide (dried over CaH2) was condensed into the resulting cholesterate solution at a temperature of -70 °C. Subsequently, the reaction mixture was allowed to warm to room temperature. The polymerization of ethylene oxide proceeded for 2 days and resulted in a light brown, highly viscous solution. The polymer was recovered by precipitation into diethyl ether. The polymer was redissolved in chloroform, and the precipitation was repeated four times to remove naphthalin and unreacted monomer. Finally, the resulting white powder was dissolved in benzene and freeze-dried. The yield of the obtained polymer was about 90%. The molecular weight of the cholesterol-substituted PEO was always in good accordance with the initial monomer-to-initiator ratio, e.g., for the described procedure 34 800 g mol-1 (with Mw/ Mn ) 1.05 using gel permeation chromatography) and a monomerto-initiator ratio of 800:1. Three grams (0.09 mmol) of this cholesterol-modified PEO was dissolved in 20 mL of dry chloroform. To this solution 1.22 g (5 mmol) of biotin, 6 mg of (dimethylamino)pyridine, and 1.04 g (5 mmol) of dicyclohexylcarbodiimide were added, and the mixture was stirred for 24 h at room temperature under an argon atmosphere. The resulting polymer was purified by repeated (8) Meier, W.; Schreiber, J. WO98/13025. (9) Constable, E. C.; Meier, W.; Nardin, C.; Mundwiler, S. Chem. Commun. 1999, 1483. (10) Lee, K.-D.; Kantor, A. B.; Nir, S.; Owicki, J. C. Biophys. J. 1993, 64, 905.
10.1021/la990915v CCC: $19.00 © 2000 American Chemical Society Published on Web 11/04/1999
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Langmuir, Vol. 16, No. 3, 2000
Notes
Table 1. Physical Data of the Chol-PEO-Bio Polymers and Results of the Aggregation of Polymer-Modified Lymphoblastoid Cells in the Presence of 50 µg mL-1 Streptavidina Mn (g mol-1) 1900 4800 9700 20 500 34 800
cmc (mol L-1) 10-6
4.5 × 3.1 × 10-6 1.3 × 10-6 6.2 × 10-7 1.5 × 10-7
A (Å2)
〈r2〉0.5 (nm)
cell aggregation
70 104 122 179 200
6 11 15 22 28
no no yes yes yes
a M , number average molecular weight; cmc, critical micelle n concentration; A, surface area occupied per molecule; 〈r2〉0.5, root mean square end-to-end distance of the PEO chains calculated assuming Gaussian statistics and excluded volume interaction.
precipitation from chloroform/diethyl ether until no more low molecular impurities could be detected. The molecular weights of the different polymers synthesized according to the procedure described above are given in Table 1. The degree of biotinylation was found to be larger than 95% by 1H NMR. Surface Tension Measurements. The surface tension γ of the various aqueous polymer solutions was determined with a Kru¨ss K8 tension balance interfacial tensiometer thermostated at 37 °C using the du-Nou¨y-ring method. The critical micelle concentration (cmc) of the polymer solutions was deduced from the discontinuity in the γ(ln cpolymer) curve. Interestingly, the cmc of the polymers decreases with increasing length of the hydrophilic PEO chain (see Table 1). This is in good agreement with recent results on very similar polymers.12 Up to now, we do not understand this behavior which is contradictory to what one would expect intuitively due to the increasing size of the watersoluble part of the molecules. The surface area occupied by one molecule was calculated using the Gibbs equation and increases with increasing molecular weight of the PEO chain. The results for the different polymers are summarized in Table 1. Cell Agglomeration. SubT1 cells (a human CD4 expressing T-lymphoblastoid cell line) were grown in suspension in RPMI medium supplemented with 10% FCS (fetal calf serum), 2 mM L-glutamine, 100 units/mL penicillin, and 100 mg of streptomycin. The number of cells in the samples was always about 106 cells/ mL of suspension. For the experiments the SubT1 cells were washed with phosphate-buffered saline (PBS), centrifuged, and subsequently taken up in a polymer solution (in RPMI medium) of the respective concentration. In the present study always a concentration of 3 g L-1 was used, i.e., the molar concentration varied from about 10-4 up to 1.5 × 10-3 mol L-1 (always above the cmc of the polymers), respectively. After addition of the polymer the cell suspensions were gently mixed and incubated for 4 h at 37 °C. To remove unbound polymer, the cells were washed again with PBS, centrifuged, taken up in RPMI medium, and plated on 35 mm plastic tissue culture dishes. To these suspensions was added a solution of streptavidin in the same buffer up to a final concentration of streptavidin of 50 mg mL-1. The samples were incubated with shaking for another 10 min at 37 °C and afterward examined directly by light microscopy.
Results and Discussion It has previously been shown that the cholesteryl end groups of respectively substituted PEOs can be anchored in the lipid membrane of vesicles or biological cells.3,13 This could be achieved in the present case by incubating the SubT1 cell cultures in the presence of the modified PEOs. From the agglutination experiments described below we have direct evidence that the Chol-PEO-Bio polymers are indeed immobilized at the cell membrane. (11) Chirovolu, S.; Walker, S.; Israelachvili, J.; Schmitt, F.-J.; Leckband, D.; Zasadzinski, J. A. Science 1994, 264, 1753. (12) Ishiwata, H.; Vertut-Doi, A.; Hirose, T.; Miyamjima, K. Chem. Pharm. Bull. 1995, 43, 1005. (13) Do¨bereiner, H.-G. Presented at the Materials Research Society Fall Meeting 1997, Boston, 1997.
However, up to now we were not able to quantify the amount of incorporated polymer. This could be done, for example, using radiolabeled polymers and will be reported in a forthcoming paper. The polymer-modified lymphoblastoid cells were treated with 50 mg mL-1 streptavidin, and the extent of agglutination was determined with a microtiter plate. The streptavidin-induced cell aggregation observed in the experiments (see Figure 1) clearly shows that an incorporation of the cholesteryl end group of the Chol-PEO-Bio polymers into the membranes of the cells was achieved. Cells which had not been treated with the polymer did not aggregate at the given streptavidin concentration. The cell aggregation is assumed to be due to ligandreceptor bonds between the biotinylated chain end of the modified PEOs and streptavidin. This is supported by the fact that the aggregation did not occur in the presence of a large excess of free, soluble biotin. Furthermore, it is well-known that ligand-receptor bonds can be reversed by the addition of an higher affinity analogue to the solution which effectively competes with and eventually replaces the originally bound ligand. To demonstrate reversibility in the present case, we added free, soluble biotin to the aggregated cells at a large excess of soluble biotin to streptavidin. It is reasonable to assume that similar to biotinylated lipids14,15 also the biotinylated polymer has a significantly lower binding affinity for streptavidin than free biotin. Indeed, after addition of an excess of biotin and incubation for about 1 h under gentle shaking of the suspension, the cells had completely redispersed back to their original state (see Figure 1). It is quite interesting to compare the effects of the PEO spacer length on the ability of the modified cells to be aggregated by the addition of streptavidin. The polysaccharide layer of the glycocalix represents a steric barrier for the protein, and hence, it has to be expected that the biotinylated end of the polymers is only accessible to the streptavidin if the PEO chain is long enough to overcome this layer. Therefore, the molecular weight of the PEO spacer chain was varied between about 2000 and 35 000 g mol-1, i.e., its root-mean-square end-to-end distance from approximately 6 up to 29 nm. Interestingly, for the polymers with Mn e 4800 g mol-1 a concentration of 50 mg mL-1 streptavidin did not induce cell agglutination, while for the polymers with Mn > 4800 g mol-1 the aggregation proceeded rapidly (see Table 1). This difference seems not to be the result of the incorporation of a lower fraction of the polymers with a lower molecular weight into the membrane. This is supported by investigations on the incorporation of cholesterylmodified PEO into lipid bilayer vesicles, which can be regarded as a model for the present system.12 Ishiwata et al.12 showed that at low concentrations of the cholesterylmodified PEOs (comparable to the present situation) the polymers are quantitatively incorporated into the lipid membranes irrespective of the molecular weight of the PEO chains. At higher concentrations a certain saturation value for the amount of incorporated polymer was observed which even increased with decreasing molecular weight of the PEO. Hence, the glycoprotein coat of the cells may be responsible for the observed difference. The biotin groups of the polymers with shorter PEO spacer chains seem to be hidden in the polysaccharide surface layer which prevents access of the relatively large and bulky protein. (14) Powers, D. D.; Willard, B. L.; Carbonell, R. G.; Kilpatrick, P. K. Biotechnol. Prog. 1992, 8, 436. (15) Green, N. M. Adv. Protein Chem. 1975, 29, 85.
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Figure 1. Photomicrographs of the reversible aggregation of SubT1 cells and schematic representation of the streptavidinmediated bridging process. The unsymmetrically substituted Chol-PEO-Bio polymers (the PEO has a chain length of n ≈ 800 in the displayed example) can be anchored in the membrane of the cells via their hydrophobic cholesteryl anchor group. The polymermodified cells can be aggregated by addition of 50 mg mL-1 streptavidin. The cell aggregates are redispersed upon addition of a large excess of free biotin. The number of cells in the samples was always about 106 cells/mL of suspension. The average diameter of one cell is approximately 10 mm.
Comparing the results with the calculated mean square end-to-end distance of the respective PEOs (see Table 1), we can conclude that the protein is not able to approach the membrane further than approximately 10-15 nm. This value is in good agreement with the typical thickness of the glycocalix of a typical cell which is about 1020 nm. Conclusions Although we have yet no quantitative data, it has qualitatively been shown that the hydrophobic anchor group of unsymmetrically substituted PEOs carrying one cholesterol and one biotin end, can be incorporated into the lipid membrane of biological cells. This is directly reflected by the fact that in contrast to the nonmodified cells the polymer-modified lymphoblastoid cells could be agglutinated by streptavidin.
Interestingly, the biotin group at the end of the PEO chain must obviously have a minimal distance of approximately 10-15 nm from the cell membrane for an effective interaction with the streptavidin molecules. This fact has been interpreted to be due to the glycocalix of the cells which prevents access of the protein to biotin groups being closer bound to the membrane surface. It has to be emphasized that the incorporation of the biotinylated polymers and, hence, the streptavidininduced aggregation seems not to be limited to lymphoblastoid cells. Investigations with other cells (e.g., hepatocytes, bacteria) are currently performed and will be reported in the future. Acknowledgment. Financial support of the Swiss National Science Foundation is gratefully acknowledged. LA990915V
