3068
Langmuir 2004, 20, 3068-3073
Physicochemical Aspects of the Liposome-Wool Interaction in Wool Dyeing Meritxell Martı´,*,† Leonid I. Barsukov,‡ Jordi Fonollosa,† Jose´ Luis Parra,† Stanislav V. Sukhanov,‡ and Luisa Coderch† Institute of Chemical and Environmental Research, IIQAB, CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain, and Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117997, Russia Received October 15, 2003. In Final Form: January 12, 2004 Despite the promising application of liposomes in wool dyeing, little is known about the mechanism of liposome interactions with the wool fiber and dyestuffs. The kinetics of wool dyeing by two dyes, Acid Green 27 (hydrophobic) and Acid Green 25 (hydrophilic), were compared in three experimental protocols: (1) without liposomes, (2) in the presence of phosphatidylcholine (PC) liposomes, and (3) with wool previously treated with PC liposomes. Physicochemical interactions of liposomes with wool fibers were studied under experimental dyeing conditions with particular interest in the liposome affinity to the fiber surface and changes in the lipid composition of the wool fibers. The results obtained indicate that the presence of liposomes favors the retention of these two dyes in the dyeing bath, this effect being more pronounced in case of the hydrophobic dye. Furthermore, the liposome treatment is accompanied by substantial absorption of PC by wool fibers with simultaneous partial solubilization of their polar lipids (more evident at higher temperatures). This may result in structural modification of the cell membrane complex of wool fibers, which could account for a high level of the dye exhaustion observed at the end of the liposome dyeing process.
Introduction Traditionally, high-temperature procedures (at about 100 °C or higher) and synthetic auxiliaries are widely used in textile dyeing. The deleterious effects of these factors are evident on both textiles (fiber damage) and the environment (contamination). These problems may be reduced by using liposomes as a natural dyeing auxiliary.1 Liposomes are vesicular structures that have an internal aqueous domain entrapped between lipid bilayers. They are formed by surface-active biological lipids; phosphatidylcholine (PC) is most commonly used for preparation of liposomes. Liposomes are of great interest as reaction microvessels and reagent carriers in various chemical and biotechnological processes. In the wool field, liposomes have been employed in the dyeing process at both laboratory and industrial levels.1,2 Earlier research on dye exhaustion, dye fixation, and textile handle has shown that liposomes can be used in wool dyeing as a vehicle alternative to synthetic textile auxiliaries. A reduction in the dyeing temperature (with corresponding savings in energy), ecological benefits (avoidance of synthetic auxiliaries), and the final quality of the textile dyed are the main advantages of the liposome dyeing process. Recently, commercial liposomes have entered the textile market as an auxiliary for liposome-assisted wool dyeing. This process is quite feasible on an industrial scale both * To whom correspondence should be addressed. Phone: (34)934006179. Fax: (34)932045904. E-mail:
[email protected]. † Institute of Chemical and Environmental Research, IIQAB, CSIC. ‡ Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences. (1) de la Maza, A.; Coderch, L.; Manich, A. M.; Bosch, P.; Parra, J. L. Nonmedical Applications of Liposomes; Barenholz, Y., Lasic, D., Eds.; CRC Press: New York, 1996; Vol. IV, p 165. (2) Coderch, L.; Martı´, M.; de la Maza, A.; Manich, A. M.; Serra, S.; Fiadeiro, J. M.; Parra, J. L. IWTO Florence Meeting, 1999; Rep. No. CTF 4.
technically and economically provided that liposomes are available at competitive prices. The application of liposomes does not require special attention and produces good results with many dyestuffs used in dyeing of pure wool and wool blends.3-5 Despite a promising start in liposome application for wool dyeing, there is still a lack of knowledge about the mechanism by which the liposomes interact with the surface of fibrous materials. Structurally, a wool fiber is an assembly of cuticle and cortical cells held together by the “cell membrane complex” (CMC). The dyeing and diffusion properties of fibers are known to be governed by this membranous structure, which is formed predominantly by internal wool lipids.6 These lipids account for only about 1.5% of total fiber weight and consist of three major lipid classes: sterols, free fatty acids, and polar lipids, predominantly ceramides, cholesterol sulfate, and glycosphingolipids. Advances have been made in characterizing this wool lipid composition7-9 and in furthering our understanding of the structure, arrangement, and thermotropic behavior of these lipid components.10,11 Internal wool lipids have been shown to form stable liposomes10,12 and are supposed to be arranged in the wool fiber as lipid bilayers. The presence of phospholipids in (3) Martı´, M.; Coderch, L.; de la Maza, A.; Manich, A. M.; Parra, J. L. Textile Res. J. 1998, 68, 209. (4) de la Maza, A.; Coderch, L.; Manich, A. M.; Martı´, M.; Parra, J. L. Textile Res. J. 1998, 68 (9), 635. (5) Martı´, M.; Serra, S.; de la Maza, A.; Parra, J. L.; Coderch, L. Textile Res. J. 2001, 71 (8), 678. (6) Leeder, J. D. Wool Sci. Rev. 1986, 63, 3. (7) Rivett, D. E. Wool Sci. Rev. 1991, 67, 1. (8) Ko¨rner, A.; Ho¨cker, H.; Rivet, D. E. Fresenius’ J. Anal. Chem. 1992, 344, 501. (9) Coderch, L.; de la Maza, A.; Soriano, C.; Erra, P.; Parra, J. L. J. Am. Oil Chem. Soc. 1995, 72, 715. (10) Coderch, L.; de la Maza, A.; Pinazo, A.; Parra, J. L. J. Am. Oil Chem. Soc. 1996, 73, 1713. (11) Fonollosa, J.; Martı´, M.; de la Maza, A.; Sabe´s, M.; Parra, J. L.; Coderch, L. Langmuir 2000, 16, 4808. (12) Ko¨rner, A.; Petrovic, S.; Ho¨cker, H. Textile Res. J. 1995, 65, 56.
10.1021/la030385+ CCC: $27.50 © 2004 American Chemical Society Published on Web 03/19/2004
Liposome-Wool Interaction in Wool Dyeing
Langmuir, Vol. 20, No. 8, 2004 3069 Table 1. Dyeing Kinetic Recipes
1A
2A
3A pretreated with liposome 1% oww
top wool sample
untreated
untreated
Acid Green 27 Acid Green 25 liposome (25 mg/mL of PC) pH (acetic acid) liquor ratio
1% owwa
1% oww
a
0.8% oww PC 4.5 1/25
4.5 1/25
4.5 1/25
1B
2B
3B
untreated
untreated
pretreated with liposome
1% oww 0.8% oww PC 4.5 1/25
1% oww
1% oww
4.5 1/25
4.5 1/25
oww: on wool weight.
Figure 1. Molecular backbone of the acid dyes used: C.I. Acid Green 27 (R1 ) C4H9) and C.I. Acid Green 25 (R1 ) CH3).
these bilayers may affect the permeability behavior of the dyes within the wool fiber. Therefore, in this work we have investigated the physicochemical interactions of liposomes with wool fibers under experimental dyeing conditions with particular interest in the liposome affinity to the fiber surface and the changes in lipid composition of the wool fibers. Correlation of these compositional and structural changes with different absorption of hydrophilic and hydrophobic dyes by the wool fibers is important for understanding the role of liposomes in the wool dyeing process. Materials and Methods South African merino top wool (21.9 µm) supplied by SAIPEL (Terrassa, Spain) was used as a dyeing textile substrate. The dyestuffs used were C.I. Acid Green 27 (hydrophobic) and C.I. Acid Green 25 (hydrophilic) (Aldrich, USA). Their chemical structures are shown in Figure 1. Partition coefficients of Acid Green 25 and 27 were obtained in a two-phase chloroform-methanol-water (1.5:1.5:0.6, v/v/v) system. The dyes were dissolved in a chloroform-methanol mixture (1:1, v/v) at a 0.2 mM concentration. The dye solution (3 mL) was mixed up with 0.6 mL of water in a Vortex mixer, and after separation of the solution in two layers, the dye content was measured in each layer by a spectrophotometric assay at 646 nm for Acid Green 25 and 648 nm for Acid Green 27. The liposomes were formed with natural soybean lecithin LIPOID S-100 (LIPOID, Switzerland) which contains about 9495% of phosphatidylcholine. Tris(hydroxymethyl) aminomethane (TRIS) was obtained from Merck. TRIS buffer was prepared as 5.0 mM TRIS adjusted to pH 7.4 with HCl, containing 100 mM of NaCl, and was filtered through Millipore membranes, type GS 0.22 µm (Bedford, MA). Liposomes of a defined size (about 200 nm) were prepared by extrusion of large unilamellar vesicles (through 800, 400, and 200 nm polycarbonate membranes) previously obtained by the reverse phase evaporation method13 to give a final concentration of 25 mg/mL of PC in TRIS buffer. The dyestuff was added to the buffer when the dye was loaded into liposomes from the aqueous phase. When the dye was incorporated into liposomes from the organic phase, the dye and the phospholipid were mixed up in an organic solvent (chloroform-methanol mixture) and the mixed lipid/dye film obtained on solvent evaporation was dispersed in the buffer. A centrifugation technique was used to separate the nonencapsulated dye (13) de la Maza, A.; Parra, J. L. Langmuir 1996, 12, 3393.
from the dye-loaded liposomes. Three to four centrifugation runs were sufficient to wash out more than 90% of the dye contained in the external aqueous medium. The vesicle size distribution and polydispersity index of liposomes after preparation and after being subjected to experimental dyeing conditions of pH and temperature were determined with dynamic light scattering measurements using a photon correlator spectrometer (Malvern Autosizer 4700c PS/MV). Samples were adjusted to the appropriate concentration range with TRIS buffer. Measurements were taken at 25 °C at a scattering angle of 90°. Experiments on liposome-wool, liposome-dye, and liposomewool-dye interactions were carried out with a standard temperature protocol: after preincubation at 40 °C, the temperature was raised at the rate of 1 °C/min to the maximum value of 90 °C and kept constant at this value for 30 min. Liposome affinity to the wool fiber was first determined for the two dyes under the usual experimental dyeing conditions. We studied the behavior of the wool previously treated with liposomes and that of the untreated wool in the presence or absence of liposomes during the dyeing process. Dyeing and liposome pretreatments were carried out in a Redchrome (Ugolini, Italy) laboratory machine, equipped with a microprocessor Becatron AG Datex-Micro (Mu¨llheim, Switzerland). Dyeing started at room temperature, and the temperature was raised at the rate of 1 °C/min until the maximum temperature (90 °C) was reached, which was maintained for 30 min. The wool samples were then rinsed with water and dried in an oven at 80 °C for 20 min. Three different dyeing processes were compared for the two dyes, Acid Green 27 (A) and Acid Green 25 (B). Wool was dyed under different experimental strategies according to the following recipes (Table 1): 1A and 1B, in the presence of liposomes; 2A, 2B, 3A, and 3B, without liposomes; 2A and 2B, without liposome pretreatment; 3A and 3B, with the wool previously subjected to liposome pretreatment under the same experimental dyeing conditions (without any dye but in the presence of liposomes, 0.8% PC owf (on weight of fibers)). Dyebath exhaustion was determined by spectrophotometry using a Shimadzu UV-265FW spectrophotometer. Dyebath liposome aliquots (0.5 mL) were periodically added to quartz cuvettes filled with 2 mL of an aqueous solution of Triton X-100 (2% w/v). The interaction between Triton X-100 and PC liposomes resulted in complete solubilization of lipids in mixed micelles,14,15 turning the liposome suspensions into a clear micellar solution. PC absorption by wool fibers during the liposome pretreatment was determined by quantitative phosphorus analysis (Phosphor Cell Test Merck, AFNOR T90-023). Complete decomposition of the sample in a solution acidified with sulfuric acid was performed by heating the cell at 120 °C in the thermoreactor TR200 (Merck) for 30 min to determine total phosphorus. Orthophosphate ions react with molybdate ions to form molybdophosphoric acid. Ascorbic acid reduces this acid to phosphomolybdenum blue, which is then determined photometrically with a Spectroquant NOVA 30 photometer (Merck). Physicochemical liposome-wool interaction was determined by differential scanning calorimetry (DSC) using dimyristoylphosphatidylcholine (DMPC; LIPOID PC 14:0/14:0, Germany) as a probe. Wool was incubated in the presence of DMPC liposomes (1% oww (on wool weight)) at different temperatures for 30 min in the 0.1 M acetate buffer (pH 4.5) containing 0.166 (14) Lichtenberg, D.; Robson, J.; Dennis, E. A. Biochim. Biophys. Acta 1983, 737, 285. (15) de la Maza, A.; Parra, J. L. Biochem. J. 1994, 303, 907.
3070
Langmuir, Vol. 20, No. 8, 2004
Martı´ et al.
Table 2. Absorption Maxima and Partition Coefficients of the Dyestuffs Selected in the Phase Chloroform-Methanol-Water System (1.5:1.5:0.6, v/v/v) dye
λmax (nm)
Kc-m-w
Acid Green 25 Acid Green 27
646 648
4.2 1.3
nomenclature AG25 AG27
M NaCl at the 1/50 liquor ratio. Incubation bath aliquots (1 mL) containing 0.2 mg/mL DMPC were taken for DSC measurements. As a control, the incubations were carried out without DMPC under the same conditions, then the aliquots (1 mL) were taken, and DMPC was added to them to the concentration of 0.2 mg/ mL. DSC measurements were carried out on a DASM-4A (Pushchino, Russia) microcalorimeter at a scan rate of 0.25 °C/ min. The broadening effect (B) was calculated from intensities of the DMPC peak at 24.6 °C according to the formula B ) (1 A/A0) × 100%, where A is a peak intensity for the experimental or control incubation, and A0 is a peak intensity for the pure DMPC standard at the concentration of 0.2 mg/mL. DMPC and internal wool lipids (IWL) liposomes used for the DSC experiments were prepared by the Vortex dispersion of dry lipid films in the 0.1 M acetate buffer (pH 4.5) containing 0.166 M NaCl at 40 and 65 °C, respectively. Internal wool lipids were Soxhlet extracted from untreated and liposome-treated wool samples with chloroform/methanol azeotrope (79:21 v/v, bath ratio 1/30) for 5 h. The lipid extracts were concentrated and stored in chloroform/methanol (2:1, v/v) at 6 °C. Aliquots were dried and weighted, and percentages of the lipid extraction were determined.9 The quantitative analysis of the samples was performed by thin-layer chromatography coupled to an automated ionization detector (TLC-FID) Iatroscan MK-5 analyzer (Iatron, Tokyo, Japan).9,10 Samples (15 µg) were spotted on Silica gel S-III Chromarods using a SES (NiederOlm, Germany) 3202/15-01 sample spotter. The rods were developed consecutively four times using the following mobile phases: (1) chloroform/methanol/water (57:12:0.6, v/v/v) twice up to the distance of 2.5 cm; (2) n-hexane/diethyl ether/formic acid (50:20:0.3, v/v/v) up to 8 cm; and (3) n-hexane/benzene (35: 35, v/v) up to 10 cm.16 This procedure was also applied to the following standards: palmitic acid and cholesterol from Merck, ceramide II from Sigma (St. Louis, MO), and phosphatidylcholine LIPOID S-100 (LIPOID, Switzerland) to determine their calibration curves for quantification of each compound.
Results and Discussion Since the main aim of this work is to determine the effects of liposomes on the transport and absorption of dyestuffs on wool fibers, two dyestuffs of known molecular structures and different sizes and hydrophilicities were selected, Acid Green 27 (AG27, hydrophobic) and Acid Green 25 (AG25, hydrophilic) (Figure 1).17,18 Absorption spectra characterization and partition coefficient measurements Kc-m-w of the two dyes were performed in the two-phase chloroform-methanol-water system modeling hydrophilic and hydrophobic environments of dye molecules in the liposome bilayer. The results obtained (Table 2) indicate the large difference between these two dyes in their affinities to the polar or nonpolar environment. The absorption spectra of these dyestuffs were recorded at temperatures of 40, 50, 60, 70, 80, and 90 °C with pH values of 4 and 5, and no differences were observed in the absorption curves. The self-assembling behavior of liposomes and their physicochemical stability at acidic pH values (4.0-5.0) and temperature range (40-90 °C) were studied by dynamic light scattering. Vesicle size and polydispersity index of bath aliquots taken at the different temperatures (16) Coderch, L.; Fonollosa, J.; Martı´, M.; Garde, F.; de la Maza, A.; Parra, J. L. J. Am. Oil Chem. Soc. 2002, 79 (12), 1215. (17) Datyner, A.; Pailthorpe, M. T. Dyes Pigm. 1987, 8, 253. (18) Pailthorpe, M. T. In Wool Dyeing; Lewis, D. M., Ed.; Publ. Society of Dyers and Colourists: Bradford, 1992; p 63.
Table 3. Vesicle Size Distribution and Polydispersity Index of Liposomes in Bath Aliquots at Different Temperatures bath sample
size (nm)
polydispersity index
room temperature 60 °C 70 °C 80 °C 90 °C 90 °C, 30 min
163.0 160.6 161.9 157.0 154.5 160.4
0.207 0.237 0.257 0.242 0.263 0.262
are presented in Table 3. These data indicate that the liposomes are stable under experimental conditions of the dyeing process. The temperatures used in the dyeing process are always higher than the transition temperature of lipids forming liposomes. This implies the continuous fluid state of these lipids maintaining the vesicles without structural modifications. Encapsulation efficiency of liposomes was studied by using two ways of dye incorporation: from the aqueous phase and from the organic phase. In the former case, an aqueous dye solution was added to the dry lipid film so that dye molecules penetrated into the inner aqueous space of liposomes at the moment of their formation. In the latter case, the lipid and the dye were mixed up first in the organic phase (chloroform-methanol mixture) and after solvent evaporation the mixed lipid-dye film was dispersed in the aqueous buffer solution. By this way, dye molecules are distributed within the lipid matrix before formation of liposomes. A comparison between these two methods of liposome loading has shown that encapsulation of acid dyes from the organic phase is more efficient than their incorporation from the aqueous phase. On loading liposomes with acid dyes from the organic phase, the saturation level was 3-4 times higher than the maximal level of the dye incorporation from the aqueous phase. This means that hydrophobic interactions contribute essentially to the binding of acid dyes to the lipid bilayer. Liposomes can influence the dyeing process through their interactions with the wool fibers and at the same time with the dyestuffs. To elucidate the effects of liposomes on each of these substrates, the dyeing kinetics for the dyes AG25 and AG27 were compared in three experimental protocols: (1) in the presence of PC liposomes, (2) without liposomes, and (3) with wool previously treated with PC liposomes. Dyeing was performed following the experimental conditions of Table 1, and the dye exhaustion was determined at different stages of the dyeing process. Results obtained for the dyes AG27 and AG25 are shown in Figure 2. For untreated wool fibers, a retarding effect of liposomes at the first stages of the dyeing process was observed in the case of the hydrophobic dye AG27 when the liposomes were present in the bath. At the end of the process, the same (AG27) or higher (AG25) dye exhaustion values were obtained when compared with the dyeing process without liposomes in the bath. At the first stages of the dyeing process, the higher retarding effect of the liposomes with the AG27 could be due to the higher affinity of the hydrophobic dye to the liposomes present in the bath in comparison with the wool fiber. In fact, the previous studies on the liposome-dye interaction and its influence on dyeing kinetics demonstrated a retarding effect on the dye exhaustion due to dye accumulation in liposomes, which takes place in measurable amounts even in the presence of wool.19 However, the most striking feature is an increase in the dye exhaustion for the two dyes at all stages of the
Liposome-Wool Interaction in Wool Dyeing
Langmuir, Vol. 20, No. 8, 2004 3071
Figure 3. Residual phosphorus in bath aliquots taken during the liposome pretreatment of wool.
Figure 2. Exhaustion kinetics of Acid Green 27 (A) and Acid Green 25 (B).
dyeing process when wool was previously treated with liposomes. The liposome-wool interaction responsible for this behavior could be explained by possible structural changes in the CMC of the fiber due to the previous PC absorption and/or the eventual wool lipid solubilization, which could increase wool permeability for the dye molecules. Therefore, additional experiments were performed to elucidate the liposome-wool interaction in the wool dyeing process. Liposome absorption by the wool fibers was followed by quantifying the amount of the total phosphorus in the bath at different stages of the liposome pretreatment. In addition, DSC measurements were carried out using DMPC liposomes as a probe to monitor changes in their thermotropic behavior that may be related to the liposome-wool interaction. Structural changes in the CMC of wool fiber were also evaluated by analyzing the lipids extracted from the liposome-treated wool fibers in order to determine whether PC is actually absorbed by the wool fibers and whether the composition of the internal wool lipids is modified. Liposome absorption by the wool fibers was determined by the quantitative total phosphorus analysis in aliquots taken from the bath under conditions of the liposome pretreatment as described in the experimental section. The results shown in Figure 3 indicate a very quick PC absorption (24%) in the first stage of the process, being also especially important in the last 30 min of incubation (19) Simonova, T. N.; Sukhanov, S. V.; Barsukov, L. I. Proceedings of the 10th International Wool Textile Research Conference, Aachen, 2000; Deutsches Wollforschungsinstitut: Aachen, Germany, 2000.
Figure 4. DSC thermograms of DMPC liposomes heated at 90 °C for 30 min in the presence or absence of wool.
at 90 °C to achieve a 39% PC absorption. If this decrease in the content of liposomes in the bath had been only due to their absorption to the wool fibers, those would contain 0.31% of PC by weight, which corresponds to about 20% of the total wool internal lipids. The influence of wool on the thermotropic properties of liposomes was studied using the DMPC liposomes as a DSC probe. Heating of the DMPC liposomes (1% oww) with wool at 70-90 °C resulted in complete disappearance of the DMPC signal from the DSC thermograms (Figure 4). Control TLC analysis and lipid phosphorus determination have shown that the incubation bath still contains 70-77% of intact DMPC. On the other hand, the light scattering data (see Table 3) have clearly brought out that liposomes remain stable under conditions of the dyeing process, retaining their integrity on long contact with wool even at high temperatures. Hence, the disappearance of the DSC signal could not be explained by extensive absorption of the DMPC liposomes to the wool fibers or by their destruction on interaction with wool. Instead, another explanation seems more plausible; viz., during high-temperature incubation, the wool may release some material that is able to interact with liposomes producing effective broadening of the DMPC peak to a baseline level. The broadening effect of wool on the DMPC peak was also observed even under milder experimental conditions at shorter incubation time and lower temperatures (Figure
3072
Langmuir, Vol. 20, No. 8, 2004
Martı´ et al. Table 4. Quantification of Main Lipid Compounds by TLC/FID
lipid extract untreated wool wool subjected to dyeing conditions wool subjected to 0.8% PC treatment under dyeing conditions
Figure 5. The DSC broadening effect observed on incubation of wool with DMPC liposomes or with buffer at different temperatures for 30 min.
Figure 6. DSC thermograms of DMPC liposomes, IWL liposomes, and a mixture of DMPC and IWL liposomes.
5). In these experiments, wool was incubated with DMPC liposomes and with the buffer containing no liposomes at temperatures from 20 to 90 °C for 30 min. After adding DMPC to the buffer experiments, the peak broadening effect in percentage was calculated (see experimental section). A distinct broadening effect was achieved already at 20 °C when wool was subjected only to the buffer treatment, but in the presence of DMPC a higher broadening effect was always observed. These results show that some liposome-active material is actually solubilized from wool, even at low temperatures, the releasing process being more efficient in the presence of liposomes. A strong effect of this solubilized material on the phase behavior of liposomes implies that this substance has a high affinity to lipid bilayers and thus may originate from the lipid constituents of the cell membrane complex of the wool fibers. In fact, the experiments performed with liposomes prepared from IWL have clearly shown that these lipids also exert a strong broadening effect on the DMPC thermogram (Figure 6). This supports our supposition that wool being incubated with liposomes releases into the incubation bath some lipid material (presumably polar lipids) that enters the liposome membrane and affects drastically its properties. A release of the lipid material from wool should be accompanied by changes in the lipid composition of wool fibers. To be certain of this, we performed a quantitative analysis of lipids extracted from the following wool
free fatty sterols acids (mg/g polar lipids phospholipid (mg/g fiber) fiber) (mg/g fiber) (mg/g fiber) 1.84 2.07
2.17 2.18
2.99 2.34
2.10
2.05
2.38
1.67
samples: (1) untreated wool fibers, (2) wool fibers subjected to the pH and temperature conditions of the dyeing process, and (3) wool fibers subjected to the liposome treatment with 0.8% PC also at pH and temperatures of the dyeing process. Lipids extracted with chloroform/methanol azeotrope were quantitatively analyzed by TLC/FID so that the main lipid families were separated and quantified. The results presented in Table 4 confirm that a decrease in the liposome content of the incubation bath observed on pretreatment of wool with liposomes (see Figure 3) is actually accompanied by the PC absorption by the wool fibers. The amount of phospholipids in the extract of wool subjected to the 0.8% PC liposome treatment accounts for 20% of PC absorbed by the fibers. This amount is lower than the maximal value of 39% obtained for the PC absorption according to the residual phosphorus analysis of the incubation bath (Figure 3). Taking into account that the wool samples were rinsed before lipid extraction, we suppose that a part of the absorbed PC was loosely bound (presumably as intact liposomes) onto the fiber surface and lost in the water rinsing. However, a significant amount of PC can be extracted from wool only with organic solvents. This means that this PC is strongly bound to the wool fibers, seemingly due to its incorporation into the lipid domains of the CMC. The amounts of free fatty acids and sterols extracted from all wool samples are very similar regardless of the experimental dyeing conditions used and the presence of liposomes in the bath. However, a substantial decrease in polar lipids was detected even when wool was subjected to the dyeing conditions in the absence of PC liposomes in the incubation bath. These experiments also show that the removal of polar lipids from wool is accompanied by simultaneous penetration of PC into the wool fibers when incubation is carried out in the presence of liposomes. Since the polar lipids consisting mainly of ceramides and cholesterol sulfate significantly differ from PC in chemical structure and membrane behavior, we suppose that such a substitution should greatly affect those properties of the CMC that govern the permeability of wool to dye molecules. Indeed, it has been shown by electron paramagnetic resonance (EPR) measurements on mixed IWL/PC liposomes that the presence of PC, especially in low amounts (10 wt %), greatly fluidizes the lipid bilayer at any temperature and decreases the enthalpy of the main phase transition of IWL from an ordered gel state to a liquidcrystalline fluid state.11 On the other hand, we have found in this work that hydrophobic interactions contribute essentially to the binding of acid dyes to the lipid bilayer. If the dye, owing to its amphiphilic nature and some affinity to the lipid bilayer, is able to diffuse along the CMC through lipid domains, then an agent that increases their fluidity will facilitate the dye penetration deep into the wool fiber. This is the effect that has been observed for the liposomepretreated wool (see Figure 2), which contains the highest amount of PC absorbed by the wool fibers. However, when
Liposome-Wool Interaction in Wool Dyeing
Langmuir, Vol. 20, No. 8, 2004 3073
the dyeing is performed in the presence of PC liposomes two different processes seem to compete against each other. On one hand, PC tends to enter the wool fibers, and on the other hand, liposomes are absorbing the dye, the latter process being more pronounced for the more hydrophobic dye. Therefore, at initial stages of the dyeing process, when the amount of PC incorporated into wool is low, the retarding effect of liposomes on the dye exhaustion kinetics predominates, which is especially obvious in the case of the hydrophobic dye AG27. The experiments described above have shown that modification of the CMC lipids with liposomes has a great influence on the dyeing and diffusion properties of wool fibers. This implies that the lipidic pathways for dye diffusion into the interior of the fiber may play a more important role in dyeing mechanisms than has hitherto been thought. Auxiliary surfactants are often used in practical dyeing processes to improve the dyeing characteristics of wool and to perform the dyeing at low temperatures. The use of liposomes offers some advantages in this respect not only because of their lower environmental impact but also due to a possibility to modify the lipid composition of wool in a controllable way by means of a partial substitution of endogenous lipids for extrinsic ones without breaking the integrity of the cell membrane complex.
of the hydrophobic dye. It appears that the hydrophobicity of liposomes competes with that of the wool fibers so that the more hydrophobic dye is retained in the dyeing bath to a greater extent. This study has also shown that liposomes and wool interact actively with each other. This interaction results in such a modification both of liposomes and wool fibers that eventually favors the dyeing process. A detailed study of the liposome-wool interaction with a variety of physicochemical methods revealed that an exchange of some lipid material between liposomes and wool fibers might occur. It was demonstrated that phosphatidylcholine from liposomes was absorbed by wool when wool fibers were subjected to the liposome treatment. On the other hand, a membrane-active factor was released from wool into the water phase, the release being highly intensified in the presence of liposomes. The strong effect it exerted on the phase behavior of liposomes implied that this material has a high affinity to lipid bilayers and may originate from the lipid constituents of the cell membrane complex of the wool fibers. This assumption was confirmed by model experiments with liposomes prepared from internal wool lipids. As far as the cell membrane complex plays a key role in penetration and diffusion of dyes into the wool fibers, these results may be helpful in a better understanding of lipidic pathways of wool dyeing.
Conclusions
Acknowledgment. This work was supported by funds from the INTAS Project 97-0487. We acknowledge the expert technical assistance of Mr. G. von Knorring.
Our findings indicate that the presence of liposomes in the dyeing bath promotes retention of the two dyes investigated, this effect being more important in the case
LA030385+
