Langmuir 1992,8, 815-823
815
Synthesis of Surface-Functionalized, Probe-Containing, Polymerized Vesicles Derived from Ammonium Bromide Surfactants C. Y. Guo, R. R. Shankar, S. H. Cai, J. Q. Wu, S. Abe, R. N. Thomas,; and J. E. Kuo Department of Chemistry, University of Missouri-Columbia, Columbia, Missouri 65211
T. Tarnowski and H. Kiang Syntex Research, Division of Syntex Corporation, Palo Alto, California 94303 Received September 6, 1991. In Final Form: December 10, 1991 A method for attaching thousands of metal ions or dye moleculesto a single protein molecule is presented. The attachment is accomplished by using a newly developed surface-functionalized polymerized vesicle derived from a mixture of monofunctionaland bifunctionalmonomers. Details of synthesisof the requisite bifunctionalmonomers, characterizationof the resulting surface-functionalized,surface-charge-controlled polymerized vesicles, and attachment of them to biomolecules are reported.
Introduction Polymerized vesicles have great potential as multisignal carriers for biomolecules, appending a great quantity of probe ions to a single immunoprotein. In one current immunoassay technique, immunoproteins are labeled with fluorescent rare earth ions such as Eu(II1) and Tb(II1) to enhance trace level detection through time-resolved fluorescence. The common way to bridge a protein with metal ions is to use a bifunctional chelating reagent such as l-@-benzenedia~onium)-EDTA'-~ or diethylenetriaminepentaacetic acid.4 However, a limitation is encountered in that an individual protein molecule allows only a few bifunctional chelating reagents to be attached to it. If too many chelating molecules are covalently bound to a host protein, the protein tends to denature, losing ita biological function. In a typical case, an IgG of molecular weight 150 OOO was labeled with 1-@-benzenediaz0nium)-EDTA. The average ratio of chelating reagent molecules to protein was less than 5. Unfortunately, this low ratio limited the utility of the bifunctional chelating reagent strategy for detection signal enhancement. It has been reported that a new chelating reagent, 4,7-bis(chlorosulfophenyl)-l,l0phenanthroline-2,9-dicarboxylicacid (BCPDA),has been incorporated into thyroglobulin at a molar ratio of 160: but the activity of the protein was not of concern. If a higher ratio is desired and protein immunoactivity is to be conserved, a new strategy must be designed. Herein is presented a method for attaching thousands of metal ions or dye molecules to a single protein molecule withcut affecting bioactivity. This is accomplished by using a newly developed surface-functionalized polymerized vesicle derived from a mixture of monofunctional and bifunctional monomers. Details of synthesis of the requisite bifunctional monomers, characterization of the
* Author to whom corresDondence should be addressed. (1) Goodwin, D. A.; Meares, C. F. Radiopharm.: Struct.-Act. Relat. [ h o c . Symp.] 1980 1981, 281-306. (2) Meares, C. F.; Wensel, T. G. Acc. Chem. Res. 1984,17, 202-209. (3) Sundberg, J. Bifunctional EDTA Analogues with Applications for the Labeling of Biological Molecules. Ph.D thesis, Stanford University, 1973. (4) Hnatowich, D. J.; Layne, W. W.; Childs, R. L. Int. J. Appl. Radiat. Isot. 1982, 33, 327-332. (5) Diamadis, E. P.; Morton, R. C.; Reichstein, E.; Khosravi, M. J. Anal. Chem. 1989, 61, 48-53.
resulting surface-functionalized, surface-charge-controlled polymerized vesicles, and attachment of them to biomolecules will be reported. Polymerized vesicle chemistry has made great progress in the last 10 years.6-12 Recent studies have shown that many surfactants which have unsaturation in their fatty chains can be polymerized under a variety of conditions.1*21 The microscopic order that is commonly present in surfactant systems places the unsaturated bonds of adjacent molecules in appropriate proximity for orderly polymerization. Free radical initiation causes a chain reaction that results in linking of the surfactant molecules at the locations of the unsaturation. In most cases, the macroscopic order of the system is not disrupted by this polymerization, which yields a microscopic "plastic bag". These systems have found applications in explosives,22 catalyst c o a t i n g ~ , 2cosmetics,26 ~-~~ the paint and coatings (6) Regen, S. L.; Czech, B.; Singh, A. J. Am. Chem. SOC. 1980,102 (21), 66384640. - - - - - - .-.
(7) Regen, S. L.; Shin, J. S.; Hainfield, J. F.; Wall, J. S. J. Am. Chem. SOC.1984,106 (19), 5756-5757. (8) Reaen, S.L.; Shin, J. S.: Yamanuchi, K. J. Am. Chem. SOC.1984. 106 (E), 5446-2447. (9) Kunitake, T.; Okahata, Y. J. Am. Chem. SOC.1977,99,3860-3861. (10) Fendler, J. H. Science 1984, 223, 888-894. (11) Tundo, P.; Kippenberger, D. J.; Klahn, P. L.; Prieto, N. E.; Jao, T.-C.; Fendler, J. H. J. Am. Chem. SOC.1982, 104,456-461. (12) Elbert, R.; Folda, T.;Ringsdorf, H. J.Am. Chem. SOC.1984,106, 7687-7692. (13)Brady, J. E.; Evans, D. F.; Kachar, B.; Ninham, B. W. J. A m . Chem. SOC. 1984, 106 (15), 4279-4280. (14) Cho, I.; Chung, K. C. Macromolecules 1984,17 (12), 2935-2937. (15) Cho, I.; Kim, C. S. Chem. Lett. 1985,10, 1543-1544. (16) Fendler, J. H. Chem. Br. 1984, 20 (12), 1098-1099, 1102-1103. (17) Fendler, J. H. Chem. Rev. 1987,85 (5), 877-899. (18) Hupfer, B.; Ringsdorf, H.; Schupp, H. Chem. Phys. Lipids 1983, 33 (4), 355-374. (19) Johnston, D. S.; Chapman, D. Liposome Technol. 1984, I , 123129. (20) Juliano, R. L.; Regen, S. L.; Singh, M.; Hsu, M. J.; Singh, A. Biol Technolom 1983.1 (10). 882-885. (21) SKgh, A.:Herendeen, B.; Gaber, B. P.; Sheridan, J. P. Polym. Mater. Sci. Eng. 1987,57, 283-285. (22) IC1 Austrailia Ltd. British Pat. GB 1314460,26 Apr 1973, Chem. Abstr. 1973, 79 (lo), 5 5 6 0 6 ~ . (23) Fendler, J.H.; Kurihara,K. Metal-Containing Polymer Systems; Plenum: New York, 1985; pp 341-353 (24) Kurihara, K.; Fendler, J. H. J. Am. Chem. SOC.1983, 105 (19), 6152-6153. (25) Hurihara, K.; Fendler, J. H.; Ravet, I.; Nagy, J. B. J. Mol. Catal. 1986, 34 (3), 325-335.
0 1992 American Chemical Society
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816 Langmuir, Vol. 8, No. 3, 1992
industry,2'g28 and p h ~ t o g r a p h y .Medical ~~ applications of polymerized vesicles in imaging,30131b i ~ a n a l y s i s ,and ~~ d r ~ g sor~multisignal ~ - ~ ~ carried9are also currently being investigated. Three unique features suggest that polymerized vesicles have the potential to act as bridges between proteins and thousands of probes: 1. The typical shelf life of the polymerized vesicles is in the order of months, during which time they display no fusion to each other. 2. Polymerized membranes of about 5 nm thickness are less permeable to ions than those of unpolymerized membranes; therefore diffusional loss of entrapped probes may be minimized. 3. The surface of a polymerizedvesicle can be chemically modified (functionalized) to promote attachment to various compounds of biomedical interest. To achieve the stated goal, three new bifunctional monomers and two new monofunctional monomers have been synthesized in our laboratory and are shown in Figure 1. The sixth monomer, containing biotin, is a derivative of one of the new monomers. The last monomer, bis[2(10-undecenoyloxy)ethylldimethylammoniumbromide, was synthesized using a published technique" but is shown here for completeness of the reactant set. The bifunctional monomers contain not only polymerizable functional groups but also biomolecule-attachment functional groups. In many of these applications, the surface charge of the polymerized vesicles is critical to the effectiveness of the product. The surface charge may determine solution stability, sensitivity to ionic strength, and adsorption onto the vesicle surface. Ammonium bromide surfactants are among the most common monomers used in these systems due to their ease of synthesis. However, a major limitation of polymerized vesicles derived from these ammonium bromide surfactants is that they carry a positive surface charge in solutions of mild pH and low ionic strength due to the partial dissociation of the bromide ions.40 A system in which the vesicle surface charge could be controlled would broaden the application of these vesicles as well as improve some systems currently in use. To form the polymerized vesicles, a monofunctional monomer and a small portion of bifunctional monomer are mixed at a ratio of about 100 to 1 (in order to limit the (26) Brown, W. H.; Vandeberg, J. T.; Poklacki, E. S.; Dachniwskj, M. J. Eur. Pat. EP209870 A2, 28 Jan 1987. Chem. Abstr. 1987, I06 (18), 143823~. (27) Gilan,J.; Kershaw,R. W. Austrailian Pat. AU445277,21Feb 1974. Chem. Abstr. 1974,Bl (4), 147894.. (28) Gunning,R. H.; Lubbock, F. J. Can. Pat. CA1033900,27 Jun 1978. Chem. Abstr. f i 7 8 , 8 9 (14), 112605~. (29) Huffey, R. F.;Lothian, D. E.; Rennison, S. C.; Titman, M. K. U S . Pat. US977002, 5 Dec 1978. Chem. Abstr. 1979, 90 (20), 160190~. (30) Gamble, R. C.; Schmidt, P. G.; Eur. Pat. EP1060552 A2, 6 Nov 1985. Chem. Abstr. 1986,105 (7), 57492~. (31) Zasadzinski, J. A. N.; Vosejpka, P. C.; Miller, W. G. J. Colloid Interface Sci. 1986, 110 (2), 347-362. (32) Hayward, J. A.; Chapman, D. Deu. Hematol. Immunol. 1986,14, 285-289. (33) Cerami, A. PCT Pat. W082/257 Al, 4 Feb 1982. Chem. Abstr. 1982, 96 (22), 187318g. (34) Freeman, F. J.; Hayward, J. A.; Chapman, D. Biochim. Biophys. Acta. 1987, 924 (2), 341-351. (35) Johnston, T. P.; Miller, S. C. J. Parenter. Sci. Technol. 1985,39 (2), 83-88. (36) Regen, S.L.; Singh, A.; Oehme, G.; Singh, M. Biochem. Biophys. Res. Commun. 1981, 101 (l),131-136. (37) Seki, K.; Tirrell, D. A.; Brand, C.; Vert, M. Makromol. Chem. 1984, 5 (4), 187-190. (38) Torchilin, V. P.; Klibanov, A. L.; Ivanov, N. N.; Ringsdorf, H.; Schlarb, B. Markomol. Chem. 1987,B (9), 457-460. (39) Chang, E. L. U S . Pat US714711AO,l6 Aug 1985. Chem. Abstr. 1986,104 (18), 155968m. (40) Bonk, F.; Hsu, M. J.; Papp, A.; Wu, K.; Regen, S. L.; Juliano, R. L. Biochim. Biophys. Acta. 1987, 900 (l),1-9.
number of functional groups on the resulting vesicle surface, an excess of which would lead to steric problems during biomolecule attachment). The polymerization is normally carried out in a solution containing metal probe ions. After polymerization, the resulting vesicle can contain thousands of metal ions inside itself without significant leakage, while presenting active groups on the outside to attach to biomolecules. The surface charge of the vesicles is easily controlled by varying the relative amounts of three types of monofunctional monomers (of negative, neutral, or positive charge) in the reaction mixture. Polymerized vesicles having surface charges ranging from strongly positive to highly negative were synthesized and characterized. The stability of suspensions of these polymerized vesicles was tested as a function of ionic strength. The adsorption properties of the vesicles as a function of the components were investigated using Bordeaux Red, salicylic acid, and Protein-A as model absorbants. Next, the coupling of surface-functionalizedvesicles to protein was accomplished. The coupling of a primary amino group of a ligand to an N-hydroxysuccinimide ester is a well understood procedure. The couplingchemistry is schematicallyshown in Figure 2. The advantage of that coupling procedure with these vesicles is that it can be carried out in aqueous solution. The vesicles are first made in a buffer, then the coupling of protein and vesicles is carried out by adding the protein to the vesicle solution. A high coupling efficiency can be expected within 2-4 h at 4 "C. Addition of the ligand causes displacement of N-hydroxysuccinimide and a stable bond is formed. The reactive ester is highly selective for primary amino groups. Biotin and avidin have become common reagents in immunochemistry due to their high affinity. They were chosen as a model here as a first step in determining if attachment to a vesicle would limit bioactivity. This probe has been detected using light, fluorescence, and electron microscope techniques. Using avidin-biotin complexes circumvents some of the problems of affinity cytochemical t e ~ h n i q u e s . ~ lThe - ~ ~high affinity constant between the glycoprotein avidin and the vitamin biotin prompted early attention to the nature of this complex. However, the innate reason for the strong interaction between biotin and avidin is not yet understood. Only the intact ureido ring in the structure of biotin is required for this strong interaction. Avidin (MM 67 000; Subunit MW 15 600; KD 10-15) is an egg white glycoprotein. The four tryptophan residues of each avidin subunit compete for the biotin molecule. In the early 19605, avidin had been used for the characterization of biotin-requiring enzymes. During the past 15 years, biotin-labeled substances have had numerous applications in cellular biochemistry. One of the major advantages of the avidin-biotin technique is that any substance which is conjugated with biotin can be detected by avidin. Unlike other immunological methods, this technique does not require an antigen-antibody reaction for detection.
-
(41) Skutelsky, E.; Bayer, E. A. Isr. J. Med. Sci. 1976, 12, 1355. (42) Singer, S. J.; Schick, A. F. J. Biophys. Biochem. Cytol. 1961, 9, 519. (43) Raff, M. C. Sci. Am. 1976,234, 30. (44) Jarrett, L.; Smith, R. M. Proc. Natl. Acad. Sci. U S A . 1975, 72, 3626.
(45) Anderson, R. G. W.; Goldstein, J. L.; Brown, M. S. Proc. Natl. Acad. Sci. U.S.A. 1976, 73, 2434. (46) Bayer, E. A.; Skutelsky, E.; Viswanatha, T.; Wilchek, M. Mol. Cell. Biochem. 1978, 19, 23. (47) Bayer, E. A.; Skutelsky, E.;Wilchek, M. Methods Enrymol. 1979, 62, 309.
Polymerized Vesicles
Langmuir, Vol. 8, No. 3, 1992 817
~3-(bromomethyl)benzyl]bis[2-(l0-undecenoyloxy)ethyl]methyl~mmonium bromide
(4-carboxybenyl)bis[2-(lO-undecenoyloxy)ethyl]methylammonium bromide
Of
N-hydroxysuccinimide ester of (4-carboxybenzyl)bis[2-(l0-undecenoyloxy~thyl]methylammonium bromide
[Bis[2-( 10-undecenoyloxy)ethyl]methylammonio]propane-3-su~onate
[Bis[2-( 1O-undecenoyloxy)ethyl]ammonio]bis(ethanesulfonate)
~(B~~~hydrazinoca~nyl)benzyl]bis[2-(10-undecenoyloxy)ethyl]methylammonium bromide
-
Br
a-Iz=CH(~&-z~z
\+p
c31z=CH(CH&rooc=HzCHz
/
kH3
Bis [ 2-( 1 0-undecenoyloxy)ethyl]dimethylammonium bromide Figure 1. Monomers used in this study.
The avidin-biotin binding is undisturbed even by extremes of pH buffer salts. This strength of avidin-biotin interaction has provided researchers with a unique tool for use in immunoassays, receptor studies, immunocytochemical staining, and protein isolation. The avidinbiotin system is particularly well suited for use in a bridging
or sandwich system, in association with antibody-antigen interactions. The biotin molecule can be easily activated and coupled to either antigens or antibodies. Subsequently, avidin can be conjugated with enzymes or fluorochromes and used as a high affinity secondary reagent which can greatly increase the sensitivity of an assay.
Guo et al.
818 Langmuir, Vol. 8, No. 3, 1992
Figure 2. Reaction for the coupling of an amine to an N-hydroxysuccinimide ester.
Experimental Section All chemicals used in the syntheses were purchased from Aldrich and used without further purification except as noted. The synthesis schemes for all monomers are shown in Figure 3. The starting materials were 10-undecenoylchloride (compound A), N-methyldiethanolamine (compound B),a,a’-dibromo-mxylene (compound E), a-bromo-p-toluic acid (compound F),N-
~FW”
zCH2 \
%=w~2h-
2CH2
+/a
/Nky-@N-N-Biotin
I I H H
Figure 3. Synthesis schemes for all monomers.
hydroxysuccinimide (compound G), and dicyclohexylcarbodiimide (compound H). Double distilled deionized water was used throughout. Bis[2-(l0-undecenoyloxy)ethyllmethylamine hydrochloride (compound C) was synthesized based on the methods reported by Fendler et al.” Synthesis of [3-(Bromomethyl)benzyl]bis[2-(1O-~ndecenoyloxy)ethyl]methylammonium Bromide. The hydrochloride acid salt (C, 2.44 g, 5 mmol) was treated with an excess of 1N sodium hydroxide (30mL) in methylene chloride (40 mL), for a pH of about 12. After solvent removal, the liquid amine (D) was added to the bromoxylene (E, 3.96g, 15 mmol) at 90 OC bath temperature under argon flow. After 3 h of reaction time, the mixture was cooled to room temperature. The product was obtained after silica gel separation. The product was a yellowish viscous liquid with a yield of about 50%. lH NMR (CDClS): a
Langmuir, Vol. 8, No. 3, 1992 819
Polymerized Vesicles
12 30 7.75-7.45 (qt, 4 H), 6.05-5.60 (m, 2 H), 5.35-5.20 (s, 2 H), 5.10: Solvent: Deionized water 4.85(m,4H),4.80-4.60(t,4H),4.60-4.50(~,3H),4.20-3.95(m, 10 - Monomer: O.ooOo25 M Excication: 298 nm 2 H), 3.50-3.20 (t, 4 H), 2.45-2.20 (t, 4 H), 2.15-1.85 (t, 4 H), 8 Emission: 402 nm 1.80-1.10 (br s, 24 H). Synthesis of (4-Carboxybenzyl)bis[2-(10-undecenoyloxy)ethyl]methylammonium Bromide. The hydrochloride - 10 salt (2.44 g, 5 mmol) was treated with 1N sodium hydroxide (30 mL) in methylene chloride (40 mL) as above. After solvent Film . Reinunder removal, the liquid amine (D) was mixed with a-bromo-p-toluic acid (F) a t a ratio of 1.00 to 1.25 mol. The reaction system was 0.0 0.5 I .o 1.5 maintained at 130 "C overnight under argon flow. The product Dye/Monomer was purified using methylene chloride. The product was a Figure 4. Adsorption of salicylic acid on polymerized vesicles. yellowish viscous solid with a melting point between 55 and 61 "C. The yield was about 55%. 'H NMR (CDCl3): 8 8.20-7.45 crystallizedfrom dimethyl ether. The melting range was between (qt, 4 H), 6.05-5.60 (m, 2 H), 5.50-5.40 (s, 2 H), 5.10-4.85 (t, 4 67 and 72 "C. The yield was slightly over 50% of the theoretical H), 4.75-4.50 (t, 4 H), 3.65-3.45 (t, 4 H), 3.00 (9, 3 H), 2.50-2.25 maximum. lH NMR (CDC13): 8 6.05-5.60 (m, 2 H), 5.10-4.85 (t, 4 H), 2.20-1.90 (t, 4 H), 1.80-1.15 (br s, 24 H). (t,4 H), 4.75-4.45 (s, 4 H), 3.85-3.25 (s, 4 H), 3.85-3.25 (br s, 8 Synthesis of N-Hydroxysuccinimide Ester of (4-CarH), 2.80-2.65 (5, 4 H), 2.50-2.20 (t, 4 H), 2.20-1.90 (m, 4 H), boxybenzyl)bis[%-(l0-undecenoyloxy)ethyl]methylammo1.80-1.15 (br s, 24 H). nium Bromide. The previous (4-carboxybenzyl)compound (0.40 Synthesis of [(4-Biotinhydrazino)carbonyl]benzylbis[2g, 0.6 mmol) in ethylene glycol dimethyl ether (10 mL) was mixed ( 10-undecenoyloxy)ethyl]met hylammonium Bromide.b2-SB with N-hydroxysuccinimide (G, 0.076 g, 0.66 mmol) in ethylene Biotin hydrazide (0.091 g, 0.35 mmol) in DMF (30 mL) wasstirred glycol dimethyl ether (4 mL) at 0 "C. Dicyclohexylcarbodiimide 10min. Then N-hydroxysuccinimideesterof (4-carboxybenzy1)(H, 0.142 g, 0.69 mmol) in ethylene glycol dimethyl ether (8mL) bis[2- (10-undecenoyloxy)ethyl]methyla"onium bromide (1.154 was added to the system. The reaction system was stirred for g, 1.5 mmol) dissolved in DMF (10 mL) was added. The reaction 48 h at 0 "C, followed by filtration to remove the precipitate, mixture was stirred for 75 h a t room temperature under an argon DCU ( d i c y c l ~ h e x y l u r e a ) .The ~ ~ ~solvent ~ was removed by a flow. After the solvent was removed, the solid product was rotovapor. The product was purified using methylene chloride. purified by ether and acetone. The melting point was measured The melting point was between 46 and 51 "C. The yield was to be 165-175 "C. about 45%. 'H NMR (CDCl3): 8 8.20-7.45 (qt, 4 H), 6.05-5.60 Formation of Vesicles. The formation of the vesicles was (m, 2 H), 5.50-5.40 (s, 2 H), 5.10-4.85 (m, 4 H), 4.65-4.50 (t, 4 performed in a 20 mL tube, 1 in. in diameter, with a ' / z in. tip H), 3.45-3.30 (t, 4 H), 3.00-2.80 (m, 7 H), 2.50-2.30 (t, 4 H), disruptor horn, using a Heat System Ultrasonic Processor (Model 2.10-1.90 (t, 4 H), 1.80-1.10 (br s, 24 H). W-225), operated at 20 kHz with a 50 W average power output Synthesis of [Bis[2-lO-undecenoyloxy)ethyl]methylamin continuous mode. The typical concentration of monomers monio]propanesulfonate. 10-Undecenoylchloride (0.22 mol) was 1to 5 mM. The ultrasonication took from 2 to 15 min to was added to (methylamino)[bis(ethanol)l (0.10 mol) in DMF transform the heterogeneous monomer solution into a clear (80 mL). The intermediate, bis[2-(l0-undecenoyloxy)ethyllhomogenized colloidal solution, colored a weak smoky blue. methylamine hydrochloride (3.96 mmol), was dissolved in methAfter ultrasonication, the vesicle solution was transferred into ylene chloride (40 mL) to which was added 1M NaOH (30 mL). a quartz tube. The polymerization was carried out by UV After the mixture was stirred overnight at room temperature, irradiation. The tube was set in the center of an RPR-100 Raythe organic layer was separated by funnel and dried with MgS04. onet Photochemical Reactor (Southern New England Ultraviolet After the solvent was removed with a rotovapor, the remaining Co.). Polymerization was not complete after 2 h, as shown by liquid amine was then placed in a three-necked flask, where it an lH NMR study, possibly due to the dynamic nature of the was mixed with 1,3-propanesultone (4.95 mmol). The system monomer layer. As described by Fendler et al.," 8-10 h under was then stirred overnight at 45 "C in an argon atmosphere. a 450-W lamp is required for complete polymerization. After cooling to room temperature, the product, [bis[2-(10-unAdsorption onto Polymerized Vesicles Having a Posidecenoyloxy)ethyl]methyla"onio]propanesulfonate, was retively Charged Surface. The adsorption onto the vesicles was crystallized from acetone and dried overnight. The white, sticky studied with Bordeaux Red (Hartman-Leddon)and salicylicacid solid was produced in a yield of slightly under 40% of the (Mallinckrodt) as model anionic compounds, and with Proteintheoretical maximum. The melting range was 141-145 "C. 'H A labeled with I25I (ICN Radiochemicals) as a model protein. NMR(CDC13): 8 605-560 (m, 2 H), 5.10-4.85 (t, 4 H), 4.75-4.45 The monomer was bis[ 2 4 l0-undecenoyloxy)ethyl]dimethylam( ~ , H), 4 4.05-3.75 (9, 6 H), 3.40-3.25 (9, 3 H), 2.95-2.80 ( ~ , H), 2 monium bromide, the positive monomer. For Bordeaux Red and 2.45-2.20 (m, 6 H), 2.20-1.90 (m, 4 H), 1.80-1.15 (br s, 24 H). salicylic acid, a series of solutions of preformed vesicles of the Synthesis of Sodium [Bis[2-( 10-undecenoyloxy)ethyl]amsame monomer concentration but different dye concentrations monio]bis[ethanesulfonate]. The starting material, 10-unwas prepared in water. The dye-to-monomer ratio ranged from decenoyl chloride (22 mmol), was mixed with [N,N-[bis-(P0.05 up to 2. Eight milliliters of each solution was forced through hydroxyethyl)]amino]ethanesulfonic acid (10 mmol) as above. an ultrafiltration membrane with a pressure of 1.5-2 atm (OmeThe resulting compound,bis[2-(l0-undecenoyloxy)ethyll(ethanegacell LKB, 10K MWCF, pore size = 2.5 nm). The filtration was su1fone)amine (0.73 mmol), was placed in a three-necked flask terminated after 4 mL of filtrate was obtained. After the ulwith condenser in an argon atmosphere. The compound was trafiltration, both the filtrate and the remainder solutions were dissolved in anhydrous trichloromethane (40 mL) containing a subjected to optical measurement (Figures 4 and 5). small volume (0.4 mL) of anhydrous pyridine. The sodium salt The detection limit of vesicles stained with Bordeaux Red was of 2-bromoethanesulfonic acid (1.825 mmol) was dissolved in determined by absorbance (Perkin-Elmer 576 ST spectrophoanhydrous DMF (45 mL) and added to the flask, and the reaction tometer) of successive dilutions of the aqueous solution. The was allowed to continue for 72 h. The color of the solution changed wavelength of maximum absorbance for vesicles stained with from colorless to yellow. The solvents were then removed at Bordeaux Red was found to be 550 mm (Figure 6). room temperature under vacuum. The sticky yellow residual The detection limit of vesicles labeled with salicylic acid was solid, sodium [bis[2-(l0-undecenoyloxy)ethyl]a"oniolbisdetermined by fluorescence emission (Perkin-Elmer MPF-3 (ethanesulfonate),was extracted with trichloromethane and re-
-
-
~~
(48) Sheehan, J. C.; Hess, G.P. J. Am. Chem. SOC.1955,77,1067-1068. (49) Sheehan, J. C.; Goodman, M.; Hess, G.P. J . Am. Chem. SOC.1956, 78, 1367-1369. (50) Sheehan, J. C.; Yang, D. D. H. J.Am. Chem. SOC.1958,80,11541158. (51) Anderson, G.W.; Zimmerman, J. E.; Callahan, F. M. J. Am. Chem. SOC.1964,86, 1839-1842.
~~
(52) Zeheb, R.; Orr, G.A. Methods Enzymol. 1986,122, 87. (53) Manz, N.; Heubner, A.; Kohler, I.;. Grill, H.; Pollow, K. J. Biochem. 1983, 131, 333. (54) Bayer, E. A.; Wilcher, M. Methods Biochem. Anal. 1980, 26, 1. (55) Hoffmann, K.; Zhang, W. J.; Romovacek, H.; Finn, F. M.; BotherBy, A. A.; Mishra, P. K. Biochemistry 1984,23, 2547. (56) Izumi, Y.; Tani, Y.; Ogata, K. Methods Enzymol. 1979,62, 326.
Guo et al.
820 Langmuir, Vol. 8,No. 3, 1992
0
-
2
1
0
Dymonomer Ratio
Figure 5. Adsorption of Bordeaux Red on polymerized vesicles. I
'
IO fiua 20 30 COhmn: Sephadex 50-150 Positive monomer ( 1 E.3 M) Diameter: 1.5 cm Rotein-A (4.3 E-9 M) Bed Volume: 1 3 ml Rotem-A Eluent Water Vesicles ( A B 0 Sample Volume: 0.3 ml
Figure 8. Elution curve of positive vesicles and Protein-A. 1003
, 0.5
I
Solvent: 0.9% NaCl UV absorption: 550 nm
0
i
B
lj -
~
-I 3
0
I
-3 -10.5
-11.0
-10.0
-9.5 -9.0 log[Vesicle]
-8.5
-8.0
6
Column: Sephadex Sa150 Dlamear: 1.5 cm Bed Volume. 13 ml Eluent Water Sample Volume: 0 3 ml
-2
-7.5
9 M'nulcs
12
15
I8
-
Vesicle(I E-3 M) Monomer 95% ncuual 5 % negauve Romin-A (4.3 E-9 M, Rorein-A Counts Vesicle Abrorbance
Figure 9. Elution curve of negative vesicles and Protein-A.
Figure 6. Detection limit of vesicles stained with Bordeaux Red.
Solvent : Deionized water Excitation: 298 nm Emission: 400 nm Slit: IO nm Sensitivity setting: 100
r
0
-
loo
I
zoo
300
400
500
600
700
Time (hours)
Figure 10. Leakage of ' T b from polymerized vesicles over time. -15
-14
.I3
-12
-11
-10
-9
-8
-7
log[vesiclel
Figure 7. Detection limit of vesicles stained with salicylic acid. fluorescence spectrophotometer) of successive dilutions of an aqueous labeled vesicle solution. Corresponding to the observed point of maximum response, the excitation wavelength was set at 298 nm and the emission wavelength at 400 nm. Both the excitation and emission slits were set at 10.0 nm. The relative fluorescence intensity was plotted against the concentration of labeled vesicles to determine the detection limit (Figure 7). Protein-A adsorption was studied using a different method. After polymerization of the vesicles, 1251-labeledProtein-A (130 pL of 4.3 nM) was added to a vesicle solution (5 mL of 1 mM). A sample (0.3mL) was loaded on a Sephadex column (Sephadex 50-150,bed volume of 13 mL, column diameter of 1.5 cm). The column was eluted with water and fractions were collected. While UV absorbance (Perkin-Elmer 576 ST spectrophotometer) was used to monitor the profile of the eluting polymerized vesicles, a y counter (Bicron 2MW212 detector, Keithley 247 high-voltage power supply, Ortec 4890 pre-amp-amp, Canberra dual counter timer 2071A) was used to monitor the eluting protein peak (Figure 8).
Adsorption onto the Surface of Vesicles Made with the Neutral a n d Negative Monomers. Vesicles of negative surface charge were prepared with 5% of the negative monomer and 95 % of the neutral monomer by the same procedure as was used for the preparation of vesicles of positive surface charge. Also, the same process of adding the three test compounds was followed for the vesicles of negative surface charge. The Protein-A study produced the results shown in Figure 9.
Probe-Containing Vesicles. The entrapment of probe ions inside the vesicle can be achieved easily by carrying out the ultrasonication in a solution of probe ions. ISgblI1 was added to the monomer solution to bring the metal ion concentration to 0.1 M. Due to the long half-life of *Sgbrrr,speed is not of the essence, although safety is. Three methods were used to separate entrapped from free probe ions: gel filtration, ultrafiltration, and dialysis.5' Among these three choices, ultrafiltration was preferred because of ita simplicity and relatively rapid speed. After the ion-containing vesicles were separated from the free ions, the vesicle solution was moved into a dialysis tube suspended in saline solution in order to study the permeability of the metal ions through the vesicle membrane. The radioactivity of both the outside and the inside of the dialysis tubing was monitored daily (Figure 10). Bonding of Protein to Surface-Functionalized Vesicles. The neutral monomer, the negative monomer, and bifunctional monomer weremixed in a 945:l ratio. The vesicles formed using this ratio had an overall negative charge and contained an active group for chemical binding. A 0.001 M vesicle solution was prepared by mixing [bis[2-(lO-undecenoyloxy)ethyllmethylammoniolpropanesulfonate (0.0115 g), bis[2-(l0-undecenoyloxy)ethyl]ammoniobis[ethanesulfonatel(0.0005g), and N-hydroxysuccinimide ester of (4-carboxybenzyl)bis[2-(l0-undecenoyloxy)ethyl]methylammonium bromide (0.0001 g). The vesicles were formed using the procedure described previously. (57)O'Brien, D.F. J. Am. Chem. SOC.1984, 106, 1627-1633.
Polymerized Vesicles
Langmuir, Vol. 8,No. 3, 1992 821 I2
I200
P
IMOT
0
IO Mlnuas 20 30 C o h n Sephadex %-IN ves~c~et ( I E-3W moMmu95% n w d . DYmCar I 5 cm 4 % negahve. 1% succmunidc w r Bed V O l 13 ~ ml Rotcm-A (4 3 E-9 M) Eluent WUQ
Figure 13. Bordeaux Red and salicylicacid molecular structures.
Sample Volume 0 3 ml
Figure 11. Bonding of protein to surface-functionalizedvesicles. -1
3
2;
\A\
20 20
-
0.20
-P-
Avidin A VIWl
-0-
Bidn-vesicles
1 0.10
aei. IO
0.00 0.0
10.0 Column: Sephadex 50-150 Bed wlume: 28 ml Eluent Phos. buffer
Minuter
20.0 Vesicles: 1 E-3 M Avidin: 3 E-9 M Sample volume: 0.15 ml
Figure 12. Attachment of avidin to the biotin-functiondized vesicles. To 5 mL of the vesicle solution, Protein-A (130 pL, 4.3 X M) was added and the mixture was allowed to stand for 6 h. A portion (0.3 mL) of this solution was loaded on a Sephadexcolumn (Sephadex 50-150, bed volume of 13mL, column diameter of 1.5 cm) and the fractions were collected (Figure 11). Attachment of Avidin to the Biotin-Functionalized Vesicles. A 0.002 M vesicle solution for polymerizing biotinylated vesicles was prepared by mixing [bis[2-(1O-undecenoyloxy)ethyl]methyla"onio]propanesulfonate (0.0115 g), [bis[2-(10-undecenoyloxy)ethyl]ammonio)bis[ ethanesulfonate] (0.0005g), and [(4-biotinhydrazinocabonyl)benzyl]bis [2- (10-undecenoy1oxy)ethyllmethylammonium bromide (0.001 g). The monomers are dissolved in a small amount of chloroform in a glass tube. The chloroform waa evaporated and phosphate buffer (pH 7.5) (10 mL, 0.02 M) was added. Polymerization was effected by the method described previously. The vesicle solution was slightly turbid. FITC-Avidin (Sigma Chemical Co.) was used without further purification. To 10 mL of the vesicle solution FITC-Avidin (0.2 mL, 1X lo-' M) was added and the solution was allowed to stand for 6 h. A portion (0.15 mL) of this solution was loaded on a Sephadex column (Sephadex 50-150, bed volume of 13 mL, column diameter of 1.5cm) and the fractions were collected. The elution profiie of the vesicles was monitored using a UV absorption detector. The fluorescenceof the avidin in the collected fraction was measured using a fluorescence spectrophotometer (MPF-3 Perkin-Elmer) (Figure 12).
Results and Discussion Polymerized vesicles of varying surface functionalization and surface charge were successfully and reproducibly formed by a relatively simple and straightforward method with reasonable yields. The vesicles showed remarkable stability in solution, both in maintaining the integrity of the sphere and in lack of leakage of entrained solution. Even partially polymerized vesicles showed a great increase in stability. It was observed that a 5 mM vesicle solution without polymerization would precipitate in a few days a t room temperature, while a partially polymerized vesicle solution stayed clear for months. Permeability of the membrane was investigated by a dialysis study after forming the vesicles in a radioactive terbium solution, entrapping approximately 4000T b ions in an average vesicle, representing a 0.1 M terbium solution inside the vesicle. Samples were taken from inside and outside of a dialysis tube at regular intervals. The
radioactive signal decay was corrected for natural radioisotope decay. Assuming permeation to be a first-order process, the diffusion constant was no more than 1.01 X h-l. Therefore, after 200 h, 2 5% or less of the T b ions were lost due to diffusional escape from the vesicles (Figure 10). Adsorption of dye moleculesonto surface-positive polymerized vesicles showed clear and reproducible results. Since both Bordeaux Red dye and salicylic acid molecules (the adsorption test probes) contain anionic groups, they presumably have a strong tendency to be adsorbed on the surface of a positive vesicle. Since the diameter of a single polymerized vesicle is about 50 nm, the vesicles could not pass through the ultrafiltration membrane used. All adsorbed dye molecules remained with the vesicles. Any free dye molecules would have passed through, since the diameters of both Bordeaux Red and salicylic acid are considerably less than the pore size of 2.5 nm. The results showed clearly that when the dye-tomonomer ratio was lower than 1.0 for Bordeaux Red (Figure 4) and 0.5 for salicylic acid (Figure 5), there were almost no free dye molecules left in the solution. The visual effect of a dark red stain being filtered to give a colorless clear solvent was quite remarkable. An electronmicrograph of polymerized vesicles stained with Bordeaux Red was made using an Hitachi H-600transmission electron spectroscope. The stained vesicles show a dark, clear-cut edge compared to the unstained vesicles (Figure 7). Each individual vesicle can adsorb 30 000 to 40 000 dye molecules or 20 OOO salicylic acid molecules in deionized water. Accordingto the dye-to-monomerratios, each probe molecule was associated with one or two monomer molecules. It is unclear at this point whether or not the dye physically penetrated the vesicle and associated with the inside lipid layer, although the terbium diffusion study makes penetration by Bordeaux Red seem unlikely. Why the Bordeaux Red, with two negative functional groups, binds twice as many molecules per vesicle as salicylic acid, which has only one, is a curiosity yet to be probed (Figure 13). Vesicles derived from substituted ammonium bromide monomers tend to act like floating ion exchange resins. The following experiments indicate that the adsorption of small ions onto the surface of the vesicles is a competitive exchange process. If the adsorption of anionic molecules onto the vesicles is due to ionic attraction, then the adsorption should be strongly influenced by the ionic strength of the solvent. When tested, solutions of high ionic strength had a tendency to release the weakly adsorbed dye. When saline (0.9% NaC1) was used as the solvent, the colloidal system became unstable. A 3000rpm centrifuge for lOmin caused settling of the stained vesicles when the dye to monomer ratio was above 0.1. However, when deionized water was used as the solvent, no such settling was observed under the same conditions.
Guo et al.
822 Langmuir, Vol. 8, No. 3, 1992
Deionized water maintains the colloidal system due to positive charge repulsion between vesicles. When the Bordeaux Red to monomer ratio was close to 1,the solution turned a little turbid (the difference in absorbance measurements from the former solution was less than 0.1 absorbance unit), but the colloidal system was still well maintained. When the dye to monomer ratio was further increased, exceeding 1, the solution again became clear, and the colloidal system was stable, apparently due to negative charge repulsion. If a dye-stained vesicle can be covalently bound to an analyte of interest, it would have excellent potential in quantitative analysis. A protein coupled to one Bordeaux M using Red-stained vesicle would be detectable at a UV-vis spectrometer (Figure 8). Salicylic acid-stained vesicles could be detected at vesicle concentration with a simple fluorescence measurement (Figure 9). If L as in a the detection volume is reduced to about capillary detection cell, detection of only mol of vesicles could be expected by a fluorometric method. This represents about 4 orders of magnitude of improvement in sensitivity compared with standard fluorescence detection using current fluorescencetagging techniques. This system works because the vesicles microscopically concentrate thousands of dye molecules. An individual vesicle can be viewed as a huge cluster of molecules. If the cross section of a monomer is taken as 0.40 nm2, each vesicle, having a diameter of about 50 nm, is made up of roughly 39 000 monomers. As the ratio of salicylic acid to monomer increased, the free bromide ions in the solution increased. This suggested that when the vesicles were suspended in deionized water, the bromide ions were physically concentrated on the surface of the vesicles. When -COO- groups, which form stronger associations with ammonium than do bromide ions, were added to the system, they displaced the bromide ions. A simple AgN03 test was made to estimate bromide concentration. After separation of the dye-stained vesicles from the solvent, a few drops of AgN03 in the filtrate caused precipitation of AgBr. By even semiquantitative determination, it was clear that the Br- concentration in the filtrate was directly related to the amount of added dye molecule. When the ionic strength was increased by use of a saline solution, the concentrated C1- displaced the bound salicylic acid due to the same ionic attraction. Each Bordeaux Red molecule contains two -SO3-groups. Acid groups in the same solution make the Bordeaux Red molecules interact more strongly with vesicles having positively charged surfaces. It was observedthat the -SO3group is adsorbed preferentially relative to the -COOgroup in that an increase in the ionic strength of the solution reduces the adsorption of salicylic acid on the membrane of the vesicle, but not of Bordeaux Red. This suggests that the association of -SO,- to the vesicle surface is much stronger than -COO-groups. Since the detection limit of fluorescence labeled vesicles is much lower than that of visible chromophores, it is highly desirable to use -SO3--containing fluorescent molecules to achieve both high sensitivity and stability. Further, once the proper ionic strength is chosen and the vesicles are saturated by the dye molecules, the nonspecific (negatively charged protein) adsorption could be minimized. Adsorption on neutral or negatively charged polymerized vesicles was also investigated. Nonspecific adsorption of protein onto positive membranes, such as was demonstrated with Protein-A, is an obstacle for the application of polymerized vesicles to in vitro immunoassay and in vivo drug delivery systems.40
I
t
rmr I
It
Figure 14. Transmission electron micrograph of surface-functionalized polymerized vesicles.
In order to minimize this surface adsorption, two newly developed monomers were used to modify the surface charge. When polymerized, the new monomers yielded vesicles of neutral and negatively charged surfaces. By adjustment of the ratio of the two monomers, the surface charge could be varied continuously. This provided a simple model to study the effect of the surface charge on the adsorption process. Even though the surface charge could be adjusted continuously by changing the monomer ratio, most of our experiments were conducted using 95% neutral monomer and 5% negative monomer. The polymerized vesicles derived from these new monomers demonstrated greatly improved surface properties. Proteins with near neutral isoelectric point (IP) show no adsorption onto the vesicle surface that was made with 5 % negative monomer (Figure 9). It should be pointed out that the aqueous solubility of the neutral monomer is less than that of the negative monomer. This is possibly due to intramolecular ion-pair formation which reduces the hydrophilic property at the head group of the monomer. Electron micrographs of the three surface-functionalized polymerized vesicles were produced using the Hitachi H-600 transmission electron microscope operated at an accelerating voltage of 100 kV. The samples of 2 mM monomer concentration were negatively stained with 2 % sodium phosphotungstate (Figure 14). The sizes of the vesicles as measured from the electron micrographs were in the range of 50 f 10 nm. This yields an average volume of a vesicle in the range of 6.5 X L, which can hold about 4000 metal ions in a single vesicle for a 0.1 M metal ion solution. Bonding of protein to surface-functionalizedvesicles has been accomplished. Figures 11and 12 show the elution profiles of the protein-vesicle systems. The retention of at least a significant part of the biotin activity
Polymerized Vesicles
was demonstrated by successful attachment of avidin to biotin-functionalizedvesicles. Unfortunately, the research ended at that point. Bioactivity experiments were performed on two similar systems of nanospheres (silica and latex) with highly successful results, but no further tests were made with these polymerized vesicles. Reports on those other two systems are in progress. Surface-functionalizedpolymerized vesicles hold great potential for the labeling of biomolecules. The functional groups of -CHZBr, -COOH, and N-hydroxysuccinimide ester can be attached to either -COOH or -NH2 groups on biomolecules. Attachment of biotin, a vitamin molecule, and avidin, a protein molecule, to the vesicles has been accomplished. This leads one to explore applications in in vivo radiotherapy, drug delivery, and in vitro immunoassay. This multi-probe-labeling method could be viewed as an adaptation of the signal amplification mechanism employed by living systems, such as hormones released from vesicles. In immunoassay, multi-probe-labeled proteins and drug metabolites using polymerized vesicles achieve a probeanalyte ratio of about 4000. This gain represents an increase of 2 to 3 orders of magnitude in sensitivity compared to the use of conventional bifunctional chelating reagents. In the related area of medical imaging, vesicles coated with proteins specific to particular tissues may be used to deliver imaging dies in great quantity to very localized sites. In radiotherapy and drug delivery, vesicles coated with proteins specific to biomolecules common to cancer or infection sites may be used to deliver radioisotopes or drugs to specific sites, greatly reducing required dosage, and, thereby, side-effects. The vesicles are small enough to pass into a body cell with its host protein. By forming vesicles with incomplete polymerization, the vesicles can be made to decompose over time.
Langmuir, Vol. 8, No. 3, 1992 823
Being hydrocarbon chains, no significant health risk is foreseen in the decomposition products. The members of this now-disbanded group invite anyone to continue this most promising work.
Acknowledgment. We are grateful to Dr. R. N. Zare for his inspiring research suggestion, to Dr. Troutner for providing us with radioisotope T b ions and facilities for radioactivity measurement, to Dr. Darrell A. Kinden at the College of Vet. Med. E.M. Facility for taking electron micrographs, and to Dr. Martin Hichens at Merck & Sharp for informative discussions. Portions of this work have been supported by a start-up fund provided by the University of Missouri-Columbia, grants from Syntex Corporation and Merck Sharp & Dohme Research Laboratories, and fellowships from ABC Laboratories and ChemChar Research. Registry No. A, 38460-95-6;B, 105-59-9;C,79919-75-8;D, 138667-33-1;E,626-15-3;F,6232-88-8;G,6066-82-6;‘ q b ,1398129-8;[3-(bromomethyl)benzyl]bis[2-(l0-undecenoyloxy)ethyl]methylammonium bromide, 138667-27-3;(4-carboxybenzy1)bis[24l0-undecenoyloxy(ethyl]methylammoniumbromide,13866728-4;(4-carboxybenzyl)bis[2-(l0-undecenoyloxy)ethyl]methylammonium bromide N-hydroxysuccinimide ester, 138667-29-5; [bis[2-(l0-undecenoyloxy)ethyllmethylammonio]propanesulfonate, 138667-30-8;sodium [bis[2-(10-undecenoyloxy)ethyl]ammoniolbis[ethanesulfonate], 138667-31-9;bis[2-(10undecenoyloxy)ethyllethanesulfone)amine,79898-74-1;sodium 2-bromoethanesulfonate,4263-52-9;[(4-biotinhydrazino)carbonyllbenzylbis[2-(l0-undecenoyloxy)ethylhyllmethylammoniumbre bis[2-(10mide, 138667-32-0;biotin hydrazide, 66640-86-6; undecenoyloxy)ethyl]dimethylammonium bromide, 79898-718; 1,3-propanesulfone,1120-71-4;[N,N-[bis(2-hydroxyethyl)]aminolethanesulfonic acid,10191-18-1;Bordeaux Red,5858-333; salicylic acid, 69-72-7.
