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Langmuir 1997, 13, 5770-5773
Cationic Polyamphiphiles in Aqueous Media: Evidence for a Fingerprint-like Structure by Cryo-Transmission Electron Microscopy A. Benjelloun, A. Brembilla,* and P. Lochon Laboratoire de Chimie-Physique Macromole´ culaire (LCPM), Unite´ associe´ e au CNRS No. 494, ENSIC-INPL, BP 451, 1 Rue Grandville, 54001 Nancy Cedex, France M. Adrian Laboratoire d’Analyse Ultrastructurale, Universite´ de Lausanne, Baˆ t. de biologie, CH-1015, Lausanne, Switzerland J. Ghanbaja Service de Microscopie Electronique en Transmission, Faculte´ des Sciences, Universite´ Henri Poincare´ , Boulevard des Aiguillettes, BP 239, 54506 Vandœuvre-le` s Nancy Cedex, France Received March 10, 1997. In Final Form: July 22, 1997
Introduction Among the variety of water-soluble polymers, the cationic amphiphilic class derived from quaternized polyheterocycles (imidazole, pyridine)1 is receiving increasing attention owing to polymer physicochemical behavior in aqueous media.2 Indeed, their properties of self-aggregation (hydrophobic microdomains)3 due to interactions between side chains give rise to the formation of a micelle-like pseudophase which can be at the root of interesting applications, for example, in favoring the dissolution,4 the transport, and the reactivity of lipophilic reactants in water.5-9 Many studies, reported in the literature, support evidence of the formation of hydrophobic microdomains in aqueous solutions of polyamphiphiles.10 In order to characterize the latter, the most widely encountered techniques are the viscosimetry, the spectroscopic methods with the use of solvatochromic probes (e.g., methylorange) through the hypsochromic shift of the long-wavelength absorption,6,7,11 and the fluorescence methods such as excited-state decay analysis and steady-state and time-resolved fluorescence spectroscopy with pyrene as a fluorescent probe.12-14 More recently, cinnamylidene type molecular rotors have been used as (1) (a) Salamone, J. C.; Israel, S. C.; Taylor, P.; Snider, B. Polymer 1973, 14, 639. (b) Salamone, J. C.; Israel, S. C.; Taylor, P.; Snider, B. J. Polym. Sci., Polym. Symp. 1974, 45, 65. (c) Salamone, J. C.; Taylor, P.; Snider, B.; Israel, S. C. Polym. Prepr., Am. Chem. Soc., Div. Polym. Chem. 1974, 15, 462. (2) Longley, K. In Surfactant Science Series, Cationic Surfactants; Jungermann, E., Ed.; Marcel Dekker: New York, 1970; Vol. 4, p 179 and references cited therein. (3) Finkelmann, H.; Jahns, E. Am. Chem. Soc., Symp. Ser. 1989, No. 384, 1. (4) Anton, P.; Laschewsky, A. Colloid Polym. Sci. 1994, 272, 1118. (5) Kunitake, T.; Shinkai, S.; Hirotsu, S. J. Org. Chem. 1977, 42, 306. (6) Shinkai, S.; Hirakawa, S. I.; Shimomura, M.; Kunitake, T. J. Org. Chem. 1981, 46, 868. (7) Wang, G. J.; Engberts, J. B. F. N. J. Org. Chem. 1994, 59, 4076. (8) Yang, Y. J.; Engberts, J. B. F. N. J. Org. Chem. 1991 56, 4300. (9) Damas, C.; Brembilla, A.; Baros, F.; Viriot, M. L.; Lochon, P. Eur. Polym. J. 1994, 30, 1215. (10) Strauss, U. P. In Microdomains in Polymer Solutions; Dubin, P. B., Ed.; Plenum Publishing Co.: New York, 1985; Vol. 51, p 1. (11) Benjelloun, A.; Damas, C.; Brembilla, A.; Lochon, P. Polym. Bull. 1994, 33, 513. (12) Binana-limbele, W.; Zana, R. Macromolecules 1987, 20, 1331. (13) Yang, J. Y.; Engberts, J. B. F. N. Recl. Trav. Chim. Pays-Bas 1991, 110, 384. (14) Winnik, F. M.; Davidson, A. R. Hamer, G. K.; Kitano, H. Macromolecules 1992, 25, 1331.
S0743-7463(97)00268-0 CCC: $14.00
Figure 1. Cryo-transmission electron micrograph of a vitrified aqueous solution of the polyamphiphile P3VPC16Br: (a, top) (concentration ) 0.5 mg/mL; magnification 150 000×), (b, bottom) (concentration ) 0.5 mg/mL; magnification 390 000×) enlargement of the selected area of Figure 1a designated by a square.
fluorescent probes in the case of polyamphiphiles bearing long alkyl-chain pyridinium units which are responsible for the pyrene quenching phenomenon.15 However, all © 1997 American Chemical Society
Notes
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Figure 2. Cryo-transmission electron micrograph of a vitrified aqueous solution of the polyamphiphile P3VPC16Br (concentration ) 1.5 mg/mL; magnification 184 000×).
these techniques provide information on the existence of microdomains only. The first tentative steps to the determination of various parameters such as size, shape, and aggregation number by using more sophisticated
means (neutron scattering,16 fluorescence quenching,11 and even transmission electron microscopy (TEM))17 remain still a matter of discussion. However, 2 decades ago, TEM by negative staining was used to get information
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on the morphological and structural properties of biomembrane models (lamellae and vesicles).18 Cryo-TEM has been further successfully applied to organized systems such as viscoelastic micellar solutions, mixed egg-yolk lethicin/conventional surfactant,19,20 and more recently polyamphiphiles.21 Nevertheless, the most common application of cryo-TEM remains the investigation of frozen hydrated biological systems (e.g., catalase, T4 and lambda bacteriophages, DNA, Semliki Forest viruses)22-24 for which this technique offers the advantage of preserving the specimens in a state close to the native one. In the present paper, we report the results of the morphology of hydrophobic microdomains of a cationic homopolymer derived from 3-vinylpyridine in aqueous medium. Experimental Section Materials. Poly(1-hexadecyl-3-vinylpyridinium bromide) (P3VPC16Br) was prepared in benzene solution by free radical polymerization of the corresponding quaternized vinylic monomer (1-hexadecyl-3-vinylpyridinium bromide) using 2,2′-azobis(isobutyronitrile) (AIBN) as an initiator. More details on the preparation of the monomer and polymerization conditions have been previously reported.11 As also described for this homopolymer,11,15 physico-chemical characterization including viscosity measurements (1-propanol-water mixtures), dye binding (with methylorange and hydrophobized methylorange), and fluorescence spectroscopy with a cinnamylidene type molecular rotor (1,1-dicyano-(4′-(dimethylamino)phenyl)-1,3-butadiene) as a fluorescent probe has revealed the formation of hydrophobic microdomains in aqueous media. Methods. Preparation of the Vitrified Hydrated Unstained Specimens. The homopolymer was previously dissolved in ethanol, then distilled water was added in such a way that the solution does not become turbid. The final ethanol concentration in the medium is 3% in volume. Before use, the solutions were allowed to stand for 24 h. The thin layer of vitrified suspension was prepared in the following manner: A 5 µL drop of the polymer solution (concentrations equal either 0.5 or 1.5 mg/mL) was deposited on a 200 mesh carbon-coated copper grid. Then the grid was held by a tweezer mounted on a guillotine-like frame and partially dried by pressing between blotting paper. The guillotine was quickly released plunging the grid in a liquid ethane container cooled to 99 K by liquid nitrogen. Care was taken to prevent formation of ice crystals (hexagonal, cubic ice layers) and/or vitreous form. The vitrified sample, kept under liquid nitrogen, was mounted on a Gatan 626 cryo-specimen holder and then rapidly introduced into the microscope (Philips CM 20). For all transfers, the temperature of the specimens was maintained at around 100 K. Because of the great sensitivity of the objects, the specimens were examined by operating in the low dose mode at an electron acceleration voltage of 120 kV in order to reduce the irradiation damage. X Microanalysis was performed by energy dispersive X-ray spectroscopy on a EDAX 9900 spectrometer. Diffraction. The image transform was determined by the use of an optical bench with laser illumination. The micrograph recorded at a magnification 45 000× acts as a diffraction grating of light, the wavelength of which is 632.8 nm (He-Ne laser). (15) Benjelloun, A.; Brembilla, A.; Lochon, P.; Adibnejad, M.; Viriot, M. L.; Carre´, M. C. Polymer 1996, 37, 879. (16) Shi, L. B.; Mauer, D. H.; Verbrugge, C. J.; Wu, C. F.; Chang, S. L.; Chen, S. H. Macromolecules 1988, 21, 3235. (17) Zhang, L.; Eisenberg, A. J. Am. Chem. Soc. 1996, 118, 3168. (18) Okahata, Y.; Tanamachi, S.; Nagai, M.; Kunitake, T. J. Colloid Interface Sci. 1981, 82, 401. (19) Talmon, Y. Colloids Surf. 1986, 19, 237. (20) Bellare, J. R.; Davis, H. T.; Scriven, L. E.; Talmon,Y. J. Electron Microsc. Tech. 1988, 10, 87. (21) Cochin, D.; Candau, F.; Zana, R.; Talmon, Y. Macromolecules 1992, 25, 4220. (22) Lepault, J.; Booy, F. P.; Dubochet, J. J. Microsc. 1983, 129, 89. (23) Lepault, J.; Gulik, A. Bull. Soc. Fr. Microbiol. 1994, 9, 191. (24) Adrian, M.; Dubochet, J.; Lepault, J.; McDowall, A. W. Nature 1984, 308, 32.
Figure 3. Laser diffraction pattern of a vitrified aqueous solution the of polyamphiphile P3VPC16Br corresponding to the area indicated by a square on the electron micrograph (Figure 2) (magnification 45 000×).
Results and Discussion Figure 1a is a Cryo-TEM micrograph (magnification 390 000) from a dilute solution of homopolymer P3VPC16Br (concentration ) 0.5 mg/mL). This print presents a tangling up of thread like objects. A more careful observation of the outer layer of these trickles shows that it is built up of a succession of black points likened to the hydrophobic microdomains, which are very close from each other. This type of organization in “strings of beads” is very comparable to that already observed by Cochin et al. in the case of poly[(vinylbenzylhexadecyldimethylammonium chloride)].21 This structure model proposed by these authors is in agreement with their investigations carried-out in time-resolved fluorescence quenching. From the print (Figure 1b), which is an elargement of a selected area of Figure 1a, it is also possible to evaluate roughly the microdomain diameter, i.e., D ) 40 ( 10 Å. The average length of a macromolecular chain (projection in the picture plane) was found to be 1200 Å but rather underestimated. Neglecting the segments linking two adjacent microdomains, the number of microdomains per macromolecular chain was estimated to be equal to about 30 for a 9.5 × 105 mol weight homopolymer.25 In these conditions, the number of alkyl chains involved in a hydrophobic microdomain would be 77. The most significant result comes from the study at a higher polymer concentration (c ) 1.5 mg/mL) for which the entanglement of strings of beads gives rise to more organized superstructures of the macromolecular chains. This organization, which has never been encountered yet for this type of polymer, was observed on micrographs with a 184 000 magnification. It has a fingerprint egg(25) Benjelloun, A. The`se de Doctorat, Institut National Polytechnique de Lorraine, NANCY, 1996.
Notes
Figure 4. Use of the tobacco mosaic virus for calibration of the microscope. Laser diffraction pattern (magnification 45 000×).
shaped form (Figure 2) in which striations, corresponding to the chains normally spaced out, can be distinguished. The striation layout is comparable to a lipidic phase image18,23 with the water layer located between the membranes. The average size of these objects is evaluated at about 500-1000 Å. With the aim of showing that we are confronted with organized structures and at optimizing the preservation of these objects during their freezing, a qualitative analysis by laser diffraction was carried out on the samples. Diffraction spots present evidence for
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the regular character of the structure (Figure 3). Indeed, the characteristics of an image can be assessed by optical diffractometry without knowing the precise frequency range of the optical diffraction pattern. For the determination of the spacing and the resolution of a specimen, the optical bench has to be calibrated using a standard spacing (in this case, an 1/10 mm graduated reticle of a magnifying-glass image was used). For the microscope, the method of calibration consists of recording the image of a standard at the same magnification (M ) 45 000) as the one used for the test images. In our case, the tobacco mosaic virus was choosen as standard. On the micrograph, only the image was examined, and thanks to a relationship between the image and the specimen structure, structural information was obtained from the image transform. The experimental resolution (given in nanometers) of a periodical structure on a Cryo-TEM print is evaluated by the measurement of the image transform diffraction pattern derived from the standard image. From Figure 4, the measurement, in reciprocal space, of the spacing between two striations of the tobacco mosaic virus structure with a spacial frequency f ) 1/2.3 (nm-1), is equal to 18.5 mm on the diffraction negative (the resolution in the real space was equal to 2.3 nm). Each millimeter on the analyzed print diffraction pattern of the polyamphiphile represents a spatial frequency f ) 1/2.3 × 18.5 (nm-1) in Fourier space. From the data of the diffraction pattern, the resolution (res ) 1/ f) of the periodical structure in the real space was estimated at 3.4 nm. Microanalysis by energy dispersive X-ray spectroscopy made was run concomitantly for elemental analysis of the sample during the observations (detection of bromine in the objects) and was suited to avoid wrong interpretations due to artifacts. At this stage of the study, it seems difficult to give a spatial picture of these structures. However, taking into account the shape of the observed objects (see Figure 2 (designed by arrows a, b)) and the striation arrangement, we could put forward as a schematic representation a regular coiling of the macromolecular chains around a spheric volume. LA970268C
