Langmuir 1999, 15, 3007-3010
Fluorescence Microscopy Observation of the Adsorption onto Hair of a Fluorescently Labeled Cationic Cellulose Ether Sudarshi T. A. Regismond,† Yew-Meng Heng,‡ E. Desmond Goddard,§ and Franc¸ oise M. Winnik*,† Department of Chemistry and Electron Microscopy Unit, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada L8S 4M1, and 849 Buccaneer Lane, Manahawkin, New Jersey 08050 Received September 3, 1998. In Final Form: January 26, 1999
Introduction Conditioning polymers are widely used in the cosmetics industry, where they are incorporated in shampoos, conditioners, and various skin lotions.1 It is generally accepted that they act by adsorbing onto the relevant keratin substrate, hair or skin. The histological structure of a hair fiber consists of three components:2,3 The outermost region, known as the cuticle, is composed of cells that form a rather thick protective coating around the cortex consisting of spindle-shaped macrofibrils. The third component, called the medulla, is a porous region in the center of the fiber. The surface of hair and of the stratum corneum of skin consists primarily of the protein R-keratin. Unaltered human hair has an isoelectric point near 3.67.4 Hence, under most pH conditions the surface of hair carries a negative charge. For this reason most conditioning polymers are cationic, since electrostatic interactions are believed to play a determining role in the adsorption mechanism. Methods for determining the amount of polymer adsorbed onto hair have been developed in several laboratories. They include direct measurement techniques, such as radiotracer analysis,5 streaming potential measurement, and ESCA analysis,6 and indirect methods, such as gel permeation chromatography.7 Although the methods may not necessarily agree on the exact amount of polymer deposited, they all concur in demonstrating without doubt that adsorption takes place. Interestingly, these experiments also gave some indications that the hair may be penetrable by a number of water-soluble agents, despite its strength, inertness, and evident “toughness”. While this fact is readily accepted for materials of low molecular weight, such as dyes,8 phenols, and waving or bleaching agents, there has been * Corresponding author. Fax: (905) 540 1310. Phone: (905) 525 9140 ext 23497. E-mail:
[email protected]. † Department of Chemistry, McMaster University. ‡ Electron Microscopy Unit, McMaster University. § 849 Buccaneer Lane, Manahawkin, NJ 08050. (1) Midha, S.; Bolich, R. E., Jr. Encyclopedia of Polymer Science and Technology; CRC Press: Boca Raton, FL, 1996; pp 2910-2916. (2) Robbins, C. R. Chemical and Physical Behavior of Human Hair, 2nd ed.; Springler Verlag: New York, 1988. (3) Swift, J. A. Fundamentals of Hair Science; Cosmetic Science Monographs 1; Micelle Press: Dorset, U.K. 1997. (4) Wilkerson, V. J. Biol. Chem. 1935, 112, 329. (5) Goddard, E. D.; Hannan, R. B.; Faucher, J. A. Proceedings of the 7th International CID Meeting, Moscow, 1976; Nats. Kom. SSSR Poverkhm.-Atzt.; Veshehestvam: Moscow, USSR; p 580. (6) Goddard, E. D.; Harris, W. C. J. Soc. Cosmet. Chem. 1987, 38, 233. (7) Blanco, B.; Durost, B. A.; Myers, R. R. J. Soc. Cosmet. Chem. 1997, 48, 127. (8) Tate, M. L.; Kamath, Y. K.; Ruetsch, S. B.; Weigmann, H.-D. J. Soc. Cosmet. Chem. 1993, 44, 347.
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some reluctance to accept the notion of penetration of higher molecular weight agents, such as certain surfactants or conditioning polymers. Scott et al.9 have shown by staining methods that certain cationic surfactants penetrate hair and that penetration increases with molecular weight. Working with wool, a closely related fiber, Griffiths and Alexander have demonstrated uptake of sodium dodecyl sulfate (SDS) of values that greatly exceed the amount required for monolayer coverage of the wool surface.10 Work on protein hydrolyzates of molecular weight 1000-10000 Da has also shown penetration into the fibers.11 Of even greater impact were the findings based on radiotracer analysis that linear poly(ethyleneimine) (Mw 600 000) is sorbed by hair to levels significantly higher than those required for monolayer coverage.12 Similar conclusions were reached by Faucher and Goddard, in their studies of the adsorption of cationic cellulose ethers.13 The latter work indicated that the amount of polymer adsorbed increased with decreasing molecular weight and that it was higher for bleached (damaged) hair than for virgin hair. Comparable data were reported for the adsorption of other cationic polymers, such as poly(dimethyldiallylammonium chloride).14,15 The current understanding of the mechanism of interaction of cationic polymers and keratin substrates invokes a diffusion process that is greatly facilitated by the substantial swelling and uptake of water that occur when hair is exposed to aqueous solutions.2 Analytical methods, which allow a visual demonstration of the presence of adsorbed polymer, can provide a view of the system, complementary to the quantitative ones already tested. Goddard and Schmitt have attempted to image polymer adsorption onto keratinic substrates by atomic force microscopy.16 Their initial tests were foiled by the lack of smoothness of natural keratin on the AFM scale. Fluorescent dyes have been used extensively in qualitative characterization of surface deposits on wool fibers17,18 and on hair.19 By scanning the fluorescence intensity along a hair fiber previously exposed to aqueous polymer solutions containing a fluorescent dye, Weigmann et al. were able to quantify the distribution of deposits on the fiber.20 In all the fluorometric studies reported to date, the dye was added to the solution and free to diffuse in solution and on the keratin surface. Since the fluorophore was not incorporated by a covalent bond into the polymer structure, the imaging experiments bring only indirect proof of the (9) Scott, G. V.; Robbins, C. R.; Barnhurst, J. D. J. Soc. Cosmet. Chem. 1969, 20, 135. (10) Griffiths, J. C.; Alexander, A. E. J. Colloids Interface Sci. 1967, 25, 311. (11) Cooperman, E. S.; Johnson, V. L. Am. Cosmet. Perfum. 1973, 88, 19. (12) Woodard, J. J. Soc. Cosmet. Chem. 1972, 23, 593. (13) Faucher, J. A.; Goddard, E. D. J. Soc. Cosmet. Chem. 1976, 27, 543. (14) Sykes, A. R.; Hammes, P. A. Drug Cosmet. Ind. 1980, 62. (15) Van Nguyen, N.; Cannell, D. W.; Mathews, R. A.; Oei, H. H. Y. J. Soc. Cosmet. Chem. 1992, 43, 259. (16) Goddard, E. D.; Schmitt, R. L. Cosmet. Toiletries 1994, 109, 55. (17) Garcia-Dominguez, J.; Julia, M. R.; de la Maza, A.; Pujol, J. M.; Sanchez, J. J. Soc. Dyers Colour. 1976, 92, 433. (18) Rothery, F. R.; White, M. A. J. Soc. Dyers Colour. 1983, 99, 11. (19) Gottschalk, H.; Hohm, G.; Kaminski, H. Proceedings of the International Wool Textile Research Conference, Aachen, 1975; Ziegler, K., Ed.; Dtsch. Wollforschungsinstitut Tech. Hochsch.: Aachen, Germany, 1976; Vol III, p 349. (20) Weigmann, H.-D.; Kamath, Y. K.; Ruetsch, S. B.; Busch, P.; Tessman, H. J. Soc. Cosmet. Chem. 1990, 41, 379.
10.1021/la9811665 CCC: $18.00 © 1999 American Chemical Society Published on Web 03/27/1999
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Notes
Figure 1. Synthetic scheme depicting the preparation of fluorescein-labeled Polymer JR400.
adsorption of the polymer itself. To eliminate this ambiguity, we have prepared a fluorescently labeled cationic cellulose ether by covalent attachment of a dye. Fluorescein was chosen as the chromophore, since its green emission is readily detected by fluorescence microscopy. Polymer JR400, a trimethylammonium chloride derivative of hydroxyethylcellulose ether was selected as polymeric substrate, as it is known to act as an effective conditioner in commercial shampoo formulations. We report here the preparation of a labeled polymer, JR400-F, (Figure 1) and a study by fluorescence microscopy of its adsorption onto hair fibers. The intensity and the patterns of fluorescent deposits along the hair surface were monitored as a function of changes in parameters, such as exposure times and post-treatment with surfactants. Experimental Section Materials. Water was deionized using a Nanopure water deionization system. 5-(4,6-Dichlorotriazinyl)aminofluorescein (5-DTAF) was purchased from Molecular Probes (Eugene, OR). Sodium dodecyl sulfate (SDS) was obtained from Sigma Chemicals. Triton-X100 was obtained from BDH Chemicals. Polymer JR400 was a gift from Amerchol Inc., Edison, NJ, and it was used without further purification. It is the chloride salt of a trimethylammonium derivative of (hydroxyethyl)cellulose with an approximate molecular weight of 400 000 Da and a level of cationic substitution of about 5 wt %. The labeled polymer JR400-F was prepared by reaction of Polymer JR400 with 5-(4,6dichlorotriazinyl)aminofluorescein21 following a procedure described for the preparation of fluorescein-labeled dextran.22 The amount of dye incorporated, determined by UV spectroscopy, was 7.4 × 10-5 mol g-1 of polymer or on average 1 dye molecule for every 75 glucose units. Analysis by gel permeation chromatography (GPC) indicated that the labeled polymer has the same molecular weight as its precursor and that all the dye was attached covalently to the polymer backbone. Methods. Fluorescence spectra were recorded at room temperature on a SPEX Fluorolog 212 spectrometer equipped with a DM3000F data system. The excitation wavelength was set at 496 nm. Emission scans were recorded from 500 to 650 nm. Gel permeation chromatography (GPC) was performed with a system consisting of a Waters 510 HPLC pump coupled to a Waters 410 differential refractometer and a Waters Tunable 486 UV-visible absorbance detector equipped with two Ultrahydrogel columns kept at 35 °C and eluted with an aqueous sodium nitrate (0.1 M) solution at a flow rate of 0.7 mL min-1. Confocal laser scanning microscopy was performed using a Zeiss LSM10 laser scanning microscope (Zeiss Ltd) equipped with a Zeiss 20× objective. A HeNe laser was used to image red emissions. An argon ion laser (488 nm) was used to image green emissions. Images were acquired with a digital Sony 3 CCD camera and processed with (21) Blakeslee, D. J. Immunol. Methods 1977, 17, 361. (22) De Belder, A. N.; Granath, K. Carbohydr. Res. 1973, 30, 375.
Figure 2. Fluorescence micrographs of a hair fiber treated with an aqueous solution of polymer JR400-F (bottom) and of a virgin hair fiber (top): hair fiber diameter, 120 µm. Northern Exposure software. Hair samples were mounted on clean glass slides using a nonfluorescing aqueous/dry mounting medium (Biomedia Corporation). They were covered with clean glass cover slips. Sample Preparation. Tresses of black hair (7.5 cm long) tied at one end were washed by a 30 min soaking in an aqueous solution of sodium dodecyl sulfate (SDS, 5%). They were rinsed thoroughly with hot tap water and several times with deionized water. They were then treated for 20 min in a saturated aqueous solution of Triton-X100. Subsequently, they were rinsed thoroughly with hot tap water and several times with deionized water. The tresses were then soaked in an aqueous solution of Polymer JR400-F (0.1 wt %, 30 mL) for either 1 min, 30 min, 12 h, or 36 h. Two samples were gathered for each soaking period. They were rinsed with deionized water for 1 min. One sample was then allowed to dry in air; the other sample was given an additional treatment with aqueous SDS (5 wt %, 10 min) and then rinsed copiously with deionized water and allowed to dry before imaging.
Results and Discussion Fluorescein-labeled Polymer JR400 was prepared by coupling of a fluoresceinyl dichlorotriazine derivative of
Notes
Figure 3. Fluorescence micrographs of a hair fiber treated with an aqueous solution of polymer JR400-F (0.1 wt %): soaking time, 12 h; top, water rinse; bottom, following treatment with aqueous SDS (5 wt %, 10 min).
fluorescein23 to Polymer JR400 in aqueous alkaline solution (Figure 1).22 The degree of substitution was kept low, to ensure that the presence of the chromophore does not alter the properties of the polymer. Through the use of gel permeation chromatography, we ascertained that the molecular weight of the labeled polymer was the same as that of the parent polymer and that the polymer was not contaminated with unreacted dye. In water Polymer JR400-F exhibits a broad featureless emission centered at 522 nm, characteristic of the fluorescein chromophore. A fluorescence micrograph of a hair fiber treated for 1 min in a 0.1 wt % Polymer JR400-F aqueous solution is presented in Figure 2 (bottom) together with a fluorescence micrograph of a virgin hair fiber (Figure 2, top). The green (23) Dudman, W. F.; Bishop, C. T. Can. J. Chem. 1968, 46, 3079.
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Figure 4. Cross sections of the top of a hair fiber treated with Polymer JR400 (0.1 wt %, 12 h): top, normal epifluorescent image; bottom, confocal fluorescent image.
fluorescence of the fluorescein label is easily detected on the fiber that was treated with the labeled polymer (bottom). It is interesting to note that the sharp demarcation of the scale edges of the cuticle is seen, suggesting enhanced deposition of the polymer between cuticular cells. This effect, which becomes more pronounced with increasing time of exposure of the hair fiber to aqueous polymer, is consistent with the known affinity of Polymer JR400 to sites of hair damage.2 A micrograph of a hair fiber soaked in aqueous JR400-F for 12 h is shown in Figure 3 (top). Next, we conducted a series of desorption studies, in which hair fibers initially treated with aqueous Polymer JR400-F for times ranging from 1 min to 12 h were washed with a solution of an anionic surfactant (SDS, 5 wt %) for 10 min. This treatment resulted in complete desorption of the polymer in the case of samples treated with the polymer for times shorter than 30 min. Such fibers
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exhibited no green fluorescence, when viewed under fluorescent light, while hair fibers immersed in pure water after treatment in aqueous solutions of JR400-F retained their original fluorescence. This confirms that the adsorbed polymer can be displaced by SDS but that it does not desorb in the absence of surfactant. Fibers soaked in water for extended periods of time could not be entirely depleted of fluorescent polymer by SDS solutions, as seen in the micrograph (Figure 3, bottom) of a fiber soaked in aqueous JR400-F for 12 h and subsequently treated with aqueous SDS. The fluorescent patterns observed prior to surfactant wash (Figure 3, top) are easily observed, albeit with significantly weaker intensity. The cross sections of the tip of a hair fiber soaked in a polymer solution for 36 h are presented in Figure 4, under normal viewing conditions (top) and under confocal imaging (bottom). In the latter case, only the plane that is in focus is observed, without interference from the layers above or below.24 This property allows imaging of structures with height differences comparable to the wavelength of the laser beam. An irregular rather thick fluorescent layer surrounds the hair surface. Whether penetration of the polymer within the hair fiber occurred cannot be ascertained from this micrograph. (24) Wilson, T. Confocal Microscopy; Academic Press: New York, 1990.
Notes
Conclusions We have investigated the adsorption of a fluorescently labeled cationic derivative of hydroxyethylcellulose onto hair fibers by laser confocal fluorescence microscopy. The method provides a visual demonstration of the deposition of polymer. It can be applied to the study of the interactions of hair and “real” shampoo formulation, thus affording a direct study of these complex colloidal systems. Moreover, it may lead to a better understanding of the mechanism of interaction of synthetic polymers and hair, as suggested also in a brief report of Swift and Allen,25 who demonstrated that, upon exposure of hair to aqueous solutions of fluorescently labeled lectins, a large order of swelling occurs in the nonkeratinous structure of hair. Acknowledgment. The work was supported by a research grant from the Natural Science and Engineering Research Council of Canada to F.M.W. LA9811665
(25) Swift, J. A.; Allen, A. K. 8th International Hair Science Symposium (Abstract) Deutches Wollforschungs Inst., Kiel, Germany, 1992.
