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Langmuir 1998, 14, 1974-1976
Charge-Induced Chromatic Transition of Amino Acid-Derivatized Polydiacetylene Liposomes Quan Cheng and Raymond C. Stevens* Department of Chemistry, University of California, and Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 Received February 13, 1998 Polydiacetylene liposomes have useful potential in biosensor research. To investigate the role of the headgroup in the colorimetric transition, a series of amino acid-derivatized 10,12-pentacosadyanoic acid lipids have been synthesized. The UV-polymerized liposomes undergo an irreversible color change from blue to red in response to a change of specific solution pH. In this system, the color change appears to be due to charge-induced headgroup rearrangement which perturbs the conjugated backbone assembly. Considering the reduced degree of freedom for lipid molecules in polymeric liposomes, a rigid, staggered conformational change in polydiacetylene conjugation is suggested as a likely mechanism for the colorimetric transition.
Liposomes composed of diacetylene-functionalized lipids have recently been shown to be effective as colorimetric biosensors.1-3 The colorimetric biosensors are self-assemblies of the diacetylene lipids mixed with natural or synthetic biological receptor molecules.2,4 Upon irradiation with short wavelength UV light, the diacetylenic lipids within the self-assemblies polymerize to form conjugated systems, giving the material a blue appearance. Perturbation of the π-orbital overlap in the conjugated system results in a blue to red color change caused by the binding of a specific biomolecule (e.g. virus or toxin).2,4 Though such utility of polydiacetylene lipid assemblies for biosensing has been established, the mechanism of color change, particularly the determinative role of the lipid headgroup, remains unclear. Recent AFM studies on polydiacetylene thin films revealed the rearrangement of the pendant side chains in the thermochromic transition.5 To investigate the charge-induced colorimetric transition, we synthesized a series of amino acid-terminated diacetylenic lipids6 (Figure 1) where charge can be manipulated in a controllable manner, aiming to better understand the function of headgroups in the polymerized liposome color transition. The mechanistic role of headgroup amino acids in promoting color transition was investigated by probing the color change of headgroup-derivatized liposomes at various pH. Using low-power (35 W) bath sonication, lipids 1-4 gave a colorless aqueous dispersion (∼1 mM) in 1-2 h. Examination by transmission electron microscopy revealed a spherical structure of liposomes with an * To whom correspondence should be addressed. (1) Reichert, A.; Nagy, J. O.; Spevak, W.; Charych, D. J. Am. Chem. Soc. 1995, 117, 829. (2) Charych, D.; Cheng, Q.; Reichert, A.; Kuziemko, G.; Stroh, M.; Nagy, J. O.; Spevak, W.; Stevens, R. C. Chem. Biol. 1996, 3, 113. (3) Pan, J. J.; Charych, D. Langmuir 1997, 13, 1365. (4) Charych, D. H.; Nagy, J. O.; Spevak, W.; Bednarski, M. D. Science 1993, 261, 585. (5) Lio, A.; Reichert, A.; Ahn, D. J.; Nagy, J. O.; Salmeron, M.; Charych, D. H. Langmuir 1997, 13, 6524. (6) Synthesis of amino acid-derivatized diacetylene lipids was based on a method described by W. Spevak. (Doctoral Thesis, University of California at Berkeley, 1993). In short, 10,12-pentacosadiynoic acid was converted to a succinimidyl ester by EDC and NHS, followed by reaction with amino acids in slightly basic THF/water solution. The product was extracted, filtered, dried, and allowed to pass through a silica gel column. Appropriate fractions were collected, and the sample was characterized by mass spectrometry. The liposomes were formed through sonication of lipids in pure water.
Figure 1. Structure of the diacetylene lipid 10,12-pentacosadiynoic acid (1) and its derivatives: lipids 2-6, amino acids derivatives; lipid 7, 3-(dimethylamino)propylamine (DMAP) derivative.
average dimension of 0.2 µm. In contrast, lipids 5 and 6, both containing hydrophobic segments in the headgroup region, did not yield liposomes even with higher powered (120 W) probe sonication. The result indicates that hydrophobic amino acids do not assemble to form an encapsulating ordering structure, unlike their hydrophilic counterparts (lipids 1-4 and cysteine/homecysteine7). Liposomes prepared from lipids 2-4 and 7 are polymerized using a UV lamp (∼254 nm) and exhibit a deep royal blue color. In comparison, the PDA lipid normally used in biosensor development (10,12-pentacosadyanoic acid, 1)1-4 forms liposomes with a less intense blue color under the same conditions. Given the initial absorption intensity of GLU-PDA liposomes as 1.00, the relative intensity for GLN-PDA, HIS-PDA, and PDA liposomes is 1.12, 1.15, and 0.24, respectively. The result suggests that amino acid derivatization ameliorates both liposome formation and color development. This observation is important in future development of colorimetric biosensors. Polymeric liposome preparations using lipids 2-4 respond to solution pH changes: increasing pH by addition (7) Neumann, R.; Ringsdorf, H. J. Am. Chem. Soc. 1986, 108, 487.
S0743-7463(98)00185-1 CCC: $15.00 © 1998 American Chemical Society Published on Web 03/28/1998
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Figure 2. Colorimetric response (CR) of liposomes for lipids 2, 3, 4, and 7 as a function of solution pH.
of NaOH resulted in an immediate blue to red transition. A quantitative analysis of the colorimetric response (CR)8 as a function of pH is shown in Figure 2. Sigmoidal curves were obtained for all three amino acid-derivatized lipids, and the CR50 values (the pH required to achieve 50% of the maximal color transition) were 6.3, 8.1, and 9.0 for GLU-PDA, HIS-PDA, and GLN-PDA, respectively. We noted that intermediate levels of color change remained stable for extended periods of time. A time-dependence experiment for GLU-PDA brought to a color transition level of CR25 (the liposomes had undergone 25% of their maximal color change at pH 6.2) revealed that the color fluctuated only 5% during the next 48 h at 4 °C. Of the three lipids, GLU-PDA is the most base sensitive, as evidenced from its low CR50 value and the steepness of its color transition slope in Figure 2. Previous studies on monolayer assemblies have suggested that the color change in polydiacetylene stems from the alkyl side chain entanglement, which leads to a shift of the π-π* electronic energy level of the conjugated backbone.9 For liposomes of lipids 2-4, we believe that the perturbation of the backbone results from surface charge interactions generated between the neighboring lipid headgroups. Repulsive Coulombic interactions develop on the surface due to ionization of the amino acid headgroups. As a result, the headgroups must rearrange themselves to accommodate the new charge distribution (staggered packing). The headgroup rearrangement thereby perturbs the lipid chains, causing a decrease in overlap of the π-orbitals within the conjugated system (Figure 3). One critical question about the PDA color change is if the general packing of the lipids in liposomes is preserved after the color transition. Chapman and co-workers (8) Colorimetric response (CR) is defined in ref 2. In this study, CR is obtained as the percent change in the adsorption at 632 nm (blue color) with respect to the total absorption at 544 nm (red color) and 632 nm. (9) Chance, R. R.; Patel, G. N.; Witt, J. D. J. Chem. Phys. 1979, 71, 206. Eckhardt, H.; Baudreaux, D. S.; Chance, R. R. J. Chem. Phys. 1986, 85, 4116. Bhattacharjee, H. R.; Preziosi, A. F.; Patel, G. N. J. Chem. Phys. 1980, 73, 1478. Lieser, G.; Tieke, B.; Wegner, G. Thin Solid Films 1980, 68, 77. Wenz, G.; Mu¨ller, M. A.; Schmidt, M.; Wegner, G. Macromolecules 1984, 17, 837. Wenzel, M.; Atkinson, G. H. J. Am. Chem. Soc. 1989, 111, 6123. Tamura, H.; Mino, N.; Ogawa, K. Thin Solid Films 1989, 179, 33. Deckert, A. A.; Horne, J. C.; Valentine, B.; Kiernan, L.; Fallon, L. Langmuir 1995, 11, 643.
Figure 3. Schematic diagram of amino acid-derivatized polydiacetylene liposomes in a chromatic transition.
reported that the structure of diacetylenic liposomes after polymerization depended on chain length and quenching temperature.10 To examine this question, GLU-PDA liposomes were tested for polymerization in basic solutions. NaOH solution was added to the liposomes followed by UV polymerization. We found that the base-treated liposomes showed no appearance of blue color following UV irradiation. Spectral analysis revealed a 94% and 98% loss of intensity at pH 7.5 and pH 9.3, respectively, as compared to that at pH 5.0. However, acidification of the base-treated solutions to pH 2.4 before UV irradiation restored a large portion of the blue color (up to 42%). The results indicate that the integrity of liposome bilayers is generally preserved under both acidic and basic conditions, but polymerization of GLU-PDA can only occur at acidic or neutral pH due to the spatial arrangement of the conjugated moieties. The inability of diacetylenic lipids to polymerize at a basic pH may be due to a reversible, staggered packing that alters the molecular distance (alignment) of the groups involved in the polymerization chemistry (Figure 3). A return to an acidic pH, however, neutralizes the headgroup charge, allowing the array to adopt a conformation that is capable of undergoing UV polymerization. The blue to red color change for polymerized liposomes was irreversible. We speculate that the headgroup ionization has a more profound impact on the side chains in the polymerized form than that on those in the unpolymerized form. Mino and co-workers reported that, at high surface pressure, a PDA monolayer on water exhibited an irreversible color change when pH was increased.11 However, a reversible color transition could be obtained when surface pressure was first released before recompression. These results implied that free movement of molecules is critical for restoration of the proper film conformation after phase transition. Unlike monolayers on an aqueous surface or unpolymerized liposomes, polymeric PDA liposomes lack such freedom of movement due to the rigid membrane structure and thus change color irreversibly. (10) Leaver, J.; Alonso, A.; Durrani, A. A.; Chapman, D. Biochim. Biophys. Acta 1983, 732, 210. (11) Mino, N.; Tamura, H.; Ogawa, K. Langmuir 1992, 8, 594.
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In contrast to the GLU-PDA- and GLN-PDA-polymerized liposomes, the HIS-PDA liposome was found to exhibit some unique properties in response to acidic pH change. Acidification of polymerized HIS-PDA liposome also results in a blue to red color change, as shown in Figure 2. Neither the GLU-PDA- or GLN-PDA-polymerized liposomes had any effect in acidic conditions. Such an acid-driven chromatic transition can be attributed to the protonation of the imidazole ring in the HIS-PDA lipid. Because the protonation of this heterocyclic ring is rather difficult, a significant amount of HCl had to be added to induce the color change. To confirm that altering the headgroup charge alone was responsible for the color transition, we tested the amine-terminated diacetylene lipid 7 (Figure 1) with addition of HCl. A reversed sigmoidal curve (7) was obtained (Figure 2), and the transition took place in an acidic medium (CR50 ) pH 4.7). These results are consistent with the charge-induced mechanism described in this paper. The charge-induced chromatic transition is predominantly due to the direct repulsive force generated at the liposome surface. Studies of the effect of ionic strength upon the chromatic transition showed that a weak color change (10% of maximal color change) for GLU-PDA liposomes could be observed when 1 M NaCl was added, indicating that high salt causes only a minor color change. Though histidine is known for its ability to coordinate metal ions, addition of 0.1 M Ni2+ to HIS-PDA liposomes did not generate any visible color change. Apparently,
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metal binding to the liposome surface does not provide enough repulsion or perturbation to the backbone. In conclusion, we have demonstrated that amino acidderivatized polymeric liposomes undergo an irreversible chromatic transition from blue to red in response to a change of solution pH. The effect appears to be due to charge-induced headgroup rearrangement which perturbs the conjugated backbone assembly. Unlike monolayers, liposomes reduce the degree of freedom for lipid rearrangement, which is required for a reversible transition, suggesting that a rigid, staggered conformational change in polydiacetylene conjugation is a likely mechanism for the colorimetric transition. Additionally, amino acid derivatization of diacetylene lipids can enhance both liposome formation and color development relative to those of the traditional diacetylene lipid and thus greatly enhance their utility as biosensor materials. Acknowledgment. This work was supported by the Director, Office of Energy Research, Office of Basic Energy Sciences, Materials Sciences Division, and the Division of Energy Biosciences of the U.S. Department of Energy, Contract No. DE-AC03-76SF0098. This work was also supported the NSF Young Investigator Award (R.C.S.) and the Beckman Foundation Young Investigator Award (R.C.S.). We thank Mark Alper for continued encouragement of this research program. LA980185B
