pubs.acs.org/Langmuir © 2010 American Chemical Society
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Loading of Curcumin in Polyelectrolyte Multilayers Paveenuch Kittitheeranun,† Neeracha Sanchavanakit,‡ Warayuth Sajomsang, and Stephan Thierry Dubas*,§ Nanoscience and Technology Program Graduate School, ‡Research Unit of Mineralized Tissues, Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand, §The Metallurgy and Materials Science Research Institute, Chulalongkorn University, Bangkok, Thailand 10330, and National Nanotechnology Center, National Science and Technology Development Agency, Thailand Science Park, Phathumthani, Thailand 12120 )
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Received January 26, 2010. Revised Manuscript Received March 6, 2010 Polyelectrolyte multilayer (PEM) thin films prepared using the layer-by-layer technique are proposed as a matrix for the immobilization of 1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6- heptadiene-2,5-dione (curcumin), a lipophilic model drug. The PEM assembly was based on the layer-by-layer deposition of cationic poly(diallyldimethyl-ammonium chloride) (PDADMAC) and anionic poly(4-styrene sulfonate, sodium salt) (PSS) onto a quartz slide. Curcumin was loaded by dipping the PEM film into a dilute solution of curcumin dispersed in an 80/20% v/v water/ethanol solution. Within a few minutes, the film turned bright yellow as a result of the curcumin loading. The effect of the solvent composition, curcumin concentration and film thickness on the final concentration of curcumin in the PEM films was measured by UV-vis spectroscopy. The loading of curcumin was driven by its partitioning in the PEM film, and its partitioning coefficient between the 80/20 solvent and the PEM thin film was found to have a value of 2.07105. The extinction coefficient of curcumin loaded into PEM was calculated to 64 000 M-1 cm-1. Results show that the loading of curcumin into the PEM films increased with the number of deposited layers, implying that curcumin partitioned into the bulk of the thin film. The maximum curcumin dose in the PEM film was measured by exposing films of various thicknesses to a high concentration (0.01% w/v) of curcumin and recording the maximum absorbance after saturation. The films thicknesses were controlled by the number of deposited PDADMAC/PSS layers (10, 20, 30, 40, 50, and 60). Results show that increasing amounts of curcumin could be loaded into the film with an increasing number of layers and up to 8 μg/cm2 of curcumin could be loaded into a 20-layer film. These results demonstrate that the loading of lipophilic curcumin in PEM thin films is done through a partitioning mechanism and that the PDADMAC/PSS film can be used as a loading matrix for lipophilic drugs
Introduction In the past few years, increasing interest has been given to the utilization of layer-by-layer self-assembly for biomedical and drugdelivery applications.1 This technique, rediscovered by Decher in the early 1990s, has been the subject of numerous reviews.2-4 This method based on electrostatic interaction between oppositely charged species involves the sequential dipping of a substrate in solutions of either polyanionic or polycationic species leading to polyelectrolyte multilayer growth. A wide range of constituents can be used in the PEM films and have been a platform of choice used in a wide range of research fields.5-7 Schlenoff, in his retrospective on the future of PEMs, suggested that PEM films will have a great future in biomedical applications such as drug and gene delivery and, more broadly, biointerfaces.8 The PEM thin films present several advantages such as tunable thickness and composition on the nanoscale. They can act as a carrier for a compound and allow its controlled release on the *Corresponding author. E-mail:
[email protected]. Tel: þ66-2-2184235. (1) Manna, U.; Patil, S. Langmuir 2009, 25, 10515–10522. (2) Lichter, J. A.; Van Vliet, K. J.; Rubner, M. F. Macromolecules 2009, 42, 8573–8586. (3) Maury, P. A.; Reinhoudt, D. N.; Huskens J. Curr. Opin. Colloid Interface Sci. 2008, 13, 74–80. (4) Ozin, G. A.; Hou, K.; Lotsch, B. V.; Cademartiri, L.; Puzzo, D. P.; Scotognella, F.; Ghadimi, A.; Thomson J. Mater. Today 2009, 12, 12–23. (5) Everett, T. A.; Higgins, D. A. Langmuir 2009, 25, 13045–13051. (6) Zan, X. J.; Su, Z. H. Langmuir 2009, 25, 12355–12360. (7) Zhuk, A.; Pavlukhina, S.; Sukhishvili, S. A. Langmuir 2009, 25, 14025– 14029. (8) Schlenoff, J. B. Langmuir 2009, 25, 14007-14010.
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basis of stimuli such as temperature,9 pH,10 and ionic strength11 as well as magnetic fields.12 Drugs and macromolecules, such as active proteins,13 enzymes,14 nucleic acids,15 and natural compounds,16 can also be immobilized into the polyelectrolyte multilayer films whereas water-soluble drugs are usually directly loaded into the PEM film through electrostatic interactions17 and lipophilic drugs are usually immobilized with ligand molecules such as modified chitosan or cyclodextrin and then integrated into the PEM film.18,19 Reported methods for the immobilization of lipophilic drugs also involve their encapsulation in nanoemulsions, liposomes, or nanocapsules as carriers that are then assembled into multilayers. Sukhishvili recently reported that the loading of small molecules and drugs was possible in hydrogenbonded PEM and displays great potential for drug-delivery (9) Shia, X.; Shenb, M.; M€ohwald, H. Prog. Polym. Sci. 2004, 29, 987–1019. (10) Dobrynina, A. V.; Rubinstein, M. Prog. Polym. Sci. 2005, 30, 1049–1118. (11) K€ostler, S.; Delgado, A. V.; Ribitsch, V. J. Colloid Interface Sci. 2005, 286, 339–348. (12) Hu, S. H.; Tsai, C. H.; Liao, C. F.; Liu, D. M.; Chen, S. Y. Langmuir 2008, 24, 11811–11818. (13) Macdonald, M.; Rodriguez, N. M.; Smith, R.; Hammond, P. T. J. Controlled Release 2008, 131, 228–234. (14) Caruso, F.; Trau, D.; M€ohwald, H.; Renneberg, R. Langmuir 2000, 16, 1485–1488. (15) Wang, L.; Yoshida, J.; Ogata, N. Chem. Mater. 2001, 13, 1273–1281. (16) Zhenqinga, H.; Zhenxia, Z.; Chuanxinb, Z.; Mei, H. J. Controlled Release 2004, 97, 467–475. (17) Fischer, D.; Dautzenberg, H.; Kunath, K.; Kissel, T. Int. J. Pharm. 2004, 280, 253–269. (18) Crespo-Biel, O.; Dordi, B.; Reinhoudt, D. N.; Huskens, J. J. Am. Chem. Soc. 2005, 127, 7594–7600. (19) Kujawa, P.; Schmauch, G.; Viitala, T.; Badia, A.; Winnik, F. M. Biomacromolecules 2007, 8, 3169–3176.
Published on Web 04/21/2010
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applications mostly because of their self-destructing and release capabilities.20,21 Although hydrogen-bonded PEM films are interesting for release applications under physiological pH, robust, nondegradable PEM films are also needed when the sustainable release of a drug is needed. Some applications such as anticancer mucoadhesive patches require a thin film that would remain stable for a long period of time for the slow release of a given drug. PEM assembled from strong polyelectrolytes such as PSS and PDADMAC is usually not though to be a good candidate for lipophilic drug loading, yet it displays very interesting properties such as fast growth under high ionic strength and chemical stability.22 In the present article, curcumin was used as a lipophilic drug and was loaded into PEM thin films. Curcumin [1,7-bis-(4hydroxy-3-methoxyphenyl)-1,6-heptadiene-2,5-dione] is a yellow pigment obtained from powdered rhizomes of Curcuma longa Linn.23 It has been used throughout history to relieve pain and accelerate wound healing in traditional medicine. Curcumin possesses chemopreventive properties and was found to exhibit antioxidative,24 anticancer,25 and anti-inflammatory activity.26 Curcumin’s poor solubility in water can be improved using a solvent mixture of ethanol and water or a surfactant such as anionic sodium dodecyl sulfate27 or cationic cetylpyridinium bromide.28 Macromolecules including gelatin,29 polysaccharides,30 poly(ethylene glycol),31 or cyclodextrins32 have also been used to stabilize curcumin in solution. Because curcumin has recently been associated with colon cancer prevention and proposed as a potent anticancer drug, our interest turned toward a study of the loading behavior of curcumin in PEM thin films for its possible used as trans-dermal drug-delivery patches based on PEM thin film technology. The scope of this study was limited to the kinetics and parameters controlling the loading of curcumin in PEM thin films. Herein, curcumin was loaded directly into PEM thin films prepared from the commonly used, non-pH-dependent PDADMAC and PSS polyelectrolytes. The loading of curcumin was studied as a function of the solvent composition, curcumin concentration, and film thickness using UV-vis spectroscopy. The extinction coefficient of curcumin loaded into the PEM film was measured, as was its partitioning coefficient. Maximum loading doses as well as loading behavior were also investigated.
Materials and Methods Chemicals and Methods. Poly(diallyldimethylammonium chloride) (PDADMAC, medium molecular weight, 20 wt % in (20) Kharlampieva, E.; Sukhishvili, S. A. Journal of macromolecular sciencew Part C: polymer reviews 2006, 46, 377–395. (21) Sukhishvili, S. A.; Kharlampieva, E.; Izumrudov, V. Macromolecules 2006, 39, 8873–8881. (22) Dubas, S. T.; Schlenoff, J. B. Macromolecules 1999, 32, 8153–8160. (23) Goel, A.; Kunnumakkara, A. B.; Aggarwal, B. B. Biochem. Pharmacol. 2008, 75, 787–809. (24) Sharma, R. A.; Gescher, A. J.; Steward, W. P. Eur. J. Cancer 2005, 41, 1955–1968. (25) Hatchera, H.; Planalpb, R.; Chob, J.; Tortia, F. M.; Tortic, S. V. Cell. Mol. Life Sci. 2008, 65, 1631–1652. (26) Jagetia, G. C.; Rajanikant, G. K. J. Surg. Res. 2004, 120, 127–138. (27) Asser, I. G. Naunyn-Schmiedeberg’s Arch. Pharmacol. 2009, 379, 47–60. (28) Lertsutthiwong, P.; Noomun, K.; Jongaroonngamsang, N.; Rojsitthisak, P.; Nimmannit, U. Carbohydr. Polym. 2008, 74, 209–214. (29) Aziz, H. A.; Peh, K. K.; Tan, Y. T. F. Drug Dev. Ind. Pharm. 2007, 33, 1263–1272. (30) Ye, S.; Wang, C.; Liu, X.; Tong, Z.; Ren, B.; Zeng, F. J. Controlled Release 2006, 112, 79–87. (31) Li, L.; Ahmed, B.; Mehta, K.; Kurzrock, R. Mol. Cancer Ther. 2007, 6, 1276–1282. (32) Tønnesen, H. H.; Masson, M.; Loftsson, T. Int. J. Pharm. 2002, 244, 127–135.
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Figure 1. Structure of curcumin used in this study. water, typical Mw = 200 000-350 000), poly(sodium 4-styrene sulfonate) (PSS, typical Mw = 70 000), and [1,7-bis-(4-hydroxy3-methoxyphenyl)-1,6-heptadiene-2,5-dione] were purchased from Aldrich, Thailand. Sodium chloride and A.R.-grade ethanol were purchased Labscan Asia Co., Ltd., Thailand. All chemicals and solvents were used as received without any further purification. Doubly distilled water was used in all experiments. PEM Thin Films. Prior to being coated with PEM films, the quartz substrates were cleaned in piranha solution (70% H2SO4/ 30% H2O2) followed by a 20 min dip in a 60 °C solution of 30% ammonia/30% H2O2/water (1:1:5 vol/vol/vol) and rinsed in distilled water. (Caution! Piranha is a strong oxidizer and should not be stored in closed containers.) The clean quartz slides were used as substrate and coated with PDADMAC and PSS by repeating the following sequence for each layer. First, the slide was dipped for 2 min into a 10 mM the polyelectrolyte solution having an ionic strength adjusted with 1 M NaCl, followed by three rinses of 1 min each in distilled water. The films were composed of 14 layers of PSS-PDADMAC except for in the experiment (Figure 7) in which the number of layers was varied (10, 20, 30, 40, 50, and 60). For the loading of curcumin in the PDADMAC/PSS PEM films, the coated slides were dipped into solutions of curcumin dispersed in a solvent with various ratios of water and ethanol. An Analytik-jena 100 UV-vis spectrophotometer was used to record the spectra of the solutions and coated thin films. An atomic force microscope from Veeco AFM (Innova) working in tapping mode with a silicon tip was used to measure the PEM film thickness.
Results and Discussion Extinction Coefficient of Curcumin in PEM Thin Films. For our study of the loading of curcumin in PEM thin films, we chose to use the PDADMAC/PSS polyelectrolyte pair because it is known to be pH-independent and stable under a wide range of condition and solvents. As seen in Figure 1, the structure of curcumin present polyphenols connected by two R,β-unsaturated carbonyl groups that provide good solubility in organic solvent such as acetone (10 mg/mL) but is poorly soluble in pure water (less than 1 mg/mL). Its loading in the PEM matrix was controlled by tuning the water/ethanol solvent composition and monitored by UV-vis spectroscopy because of its strong absorbance in the visible range (λmax = 430 nm). As seen in Figure 2, upon dipping of a 14-layer PDADMAC PSS PEM film in a 0.00025% curcumin solution of 80/20% water/ethanol, a yellow color appeared on the film as a result of curcumin loading. The kinetics of loading can be plotted as a function of time and was found to increase sharply and stop when equilibrium was reached. Because our work involved the quantification of the amount of curcumin present both in the release solutions and in the PEM films, it was necessary to recalculate the extinction coefficient (ε20/80, εpem) of curcumin in each medium. The release solution contains mostly ethanol and is used to release the curcumin loaded into the PEM film. Values of ε for curcumin are available in databases but vary depending on the solvent used and the surrounding matrix. ε20/80 corresponds to the extinction coefficients of curcumin in the 20/80% water/ethanol solvent whereas εpem corresponds to extinction coefficients of curcumin diffused in Langmuir 2010, 26(10), 6869–6873
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Figure 2. Absorbance values at λmax as a function of time for a 14-layer PEM film dipped into a 0.00025% w/v solution of curcumin. The corresponding absorbance spectra are compiled in the cartouche. Pictures of the sample taken during the loading process are also shown.
Figure 3. Calibration curve of curcumin in 20/80% water/ethanol. The calculated value for the extinction coefficient, ε20/80, is 75 969.
the PEM film. εpem is needed to convert the absorbance values from curcumin loaded into the PEM film into concentration values using Beer’s law. First, ε20/80 was calculated from the slope of the calibration curve between the absorbance for curcumin concentrations of 0.01, 0.05, 0.1, 0.5, and 1 ppm. The UV-vis spectra for each concentration and the final calibration curve are shown in Figure 3, and the extinction coefficient value of ε20/80 = 75969 M-1 cm-1 was obtained Second, εpem of curcumin in the PEM film was calculated. This can be done by loading a PEM film with curcumin and recording its absorbance as seen in plot A of Figure 4. Then the curcumin loaded into the film is released in a good solvent for curcumin such as 20/80 water/ethanol as seen in plot B Figure 4. The absorbance of the obtained solution is converted from absorbance to concentration and finally to moles, which were initially present in the film. If the thickness and surface of the film are known, it is then possible to calculate the concentration, and using Beer’s law, Langmuir 2010, 26(10), 6869–6873
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Figure 4. Absorbance spectra of (A) a 14-layer PSS/PDADMAC film loaded with curcumin and (B) a 50 mL release solution of 20/80% water/ethanol used to dissolve the curcumin in sample A.
the extinction coefficient of curcumin in the film can be extracted. High ethanol content is used to wash out all of the curcumin in the film, yet the 20/80% water/ethanol solvent was preferred over 100% ethanol because it was found to be more efficient for the release of curcumin from the PEM film as a result of a better interaction with the PEM matrix. Atomic force microscopy was used to measure the PEM film thickness by making a scratch on the glass slide with a sharp needle and measuring the step height. Looking then at the cross section of the 14-layer PSS/PDADMAC film, a thickness of 130 nm was measured, which is in accordance with previously reported data. Finally, for a film surface of 2.5 cm 3.5 cm and a thickness of 130 nm, the final extinction coefficient of curcumin in PEM films was εpem = 64 000 M-1 cm-1. The εpem of curcumin is lower than ε20/80 because of the different electrostatic environment as well as some possible π-π interactions between the curcumin and PSS aromatic structures. Lower extinction coefficients for curcumin were previously reported for curcumin dispersed in a PVA hydrogel with ε = 24 000 M-1 cm-1.33 It can also be seen that there is a λmax shift of 10 nm between the absorbance spectra of curcumin in solution and in the PEM film. This can be due to the stacking of the curcumin molecule in aggregates, which would perturb the electronic state of the molecule, leading to such a λmax shift. Solvent Effect. The loading of curcumin as a neutral hydrophobic drug is driven by a partitioning mechanism in which PEM plays the role of the hydrophobic phase and the loading solvent is the hydrophilic phase. The effect of solvent composition on the loading of curcumin in the PEM film is shown in Figure 5. When curcumin was dissolved in pure ethanol, no loading in the PEM film was observed (data not shown) because of its good solubility in this solvent. Nevertheless, when the volume fraction of water in ethanol was increased to above 50%, the partitioning of curcumin in the PEM film was visible as the film turned bright yellow. Although PEM films are prepared from charged polyelectrolytes and are known to be hydrophilic, the polyelectrolyte complexes that are the building blocks of the PEM thin films are insoluble (33) Shah, C. P.; Mishra, B.; Kumar, M.; Priyadarsini, K. I.; Bajaj, P. N. Curr. Sci. 2008, 95, 1426–1432.
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Figure 7. Plot of the calculated concentration of curcumin partitioned in a PEM film as a function of the curcumin concentration in the loading solvent (80/20% v/v water/ethanol).
Figure 5. Absorbance spectra of curcumin in PEM thin films loaded from 0.00025% curcumin in solutions of various water/ ethanol ratios.
Figure 6. Absorbance of curcumin loaded into PDADMAC/PSS films with various top layer charges and numbers of layers.
and the resulting structure possess a rather hydrophobic character. Although the region near the surface of the PEM is highly charged, the bulk of the PEM is neutral and offers a favorable environment for lipophilic molecules as long as they can diffuse through the top electrostatic layer. Shown in Figure 6 is the absorbance of a film loaded with curcumin when the charge of the top layer is either positive (PDADMAC) or negative (PSS). It can be seen that the final absorbances are nearly equal, independent of the film surface charge, and that neutral curcumin can readily diffuse through the mixing zone and diffused into the bulk of the film. Justifications for the bulk diffusion versus surface absorption are discussed in the last paragraph. Partition Coefficient. Because the loading of neutral molecules is driven by a partitioning mechanism resulting from favorable thermodynamic interactions, the partition coefficient (K) of curcumin in PEM films was measured. The K value is measured at equilibrium by taking the ratio of the final concentration of a compound between two immiscible polar and nonpolar solvents, 6872 DOI: 10.1021/la1003676
Figure 8. UV-vis absorbance of curcumin loaded into PEM thin films with an increasing number of deposited layers as a function of time.
usually octanol and water. In our study, the PEM thin film was used as the nonpolar phase, and a mixture of water and ethanol was used as the polar phase. The choice of 80/20% water/ethanol as a solvent was made because it led to strong partitioning of curcumin in the PEM and provides good stability of the curcumin against photodegradation. Higher ethanol content displayed low partitioning of curcumin in the film whereas curcumin light stability was poor for solvents with higher water contents. When a PEM thin film was dipped into a 80/20% water/ethanol solution containing 0.00025% curcumin, partitioning of curcumin occurred. Although curcumin is soluble in this solvent, the PEM appears to be more hydrophobic and curcumin starts to diffuse within the PEM film. To measure the partition coefficient, this experiment is repeated for various curcumin concentrations (0.001, 0.0005, 0.00025, 0.0001, and 0.00005% w/v) and the film absorbance measured after equilibrium was reached. Shown in Figure 7 is a plot of the relationship between curcumin concentration in the solvent and curcumin concentration in the PEM. The concentration of curcumin in the PEM film was converted from absorbance measurements using the extinction Langmuir 2010, 26(10), 6869–6873
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coefficient εpem calculated in the previous paragraph. From the slope of the fitting curve a partitioning value of K = 1.25 105 was then calculated for the 80/20 water solvent. A value of 3.66 105 has been reported for curcumin partitioning between the aqueous phase and CTAB micelles, which is expected to be higher because of the high hybrophobicity of the alkyl chains.34 Maximum Curcumin Dose in the Film. Because the application of this research could be the development of a nanocoating used as patch for transdermal drug delivery, it is important to know the maximum loading or dose of curcumin available for delivery from the PEM films for a given thickness. Using the calculated extinction coefficient εpem, direct quantification of the curcumin content in the film expressed in g/cm2 was then possible. It is also interesting to know whether the partitioning of curcumin occurs only at the surface or in the bulk of the PEM film. In Figure 8, the absorbance values of curcumin as a function of time in the PEM film of increasing thickness are compiled. A solution of 0.01% w/v curcumin was used as the loading solution, which is quite concentrated and leads to the saturation of curcumin in the film. It can be seen that the absorbance value at 440 nm increases before reaching a plateau confirming the saturation of the PEM layers with the curcumin in solution. The total curcumin content in the film was found to increase linearly with the number of deposited layers as seen on Figure 8. A linear relationship between the total amount of loaded curcumin and the number of layers implies that curcumin diffuses in the bulk of the PEM and does not simply adsorb at the surface of the film. If this was the case, then the amount of curcumin would have been independent of the number of layers and remained constant. The absorbance (34) Iwunze, M. O. J. Mol. Liq. 2004, 111, 161–165.
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converted into microgram of curcumin per cm2 shows a maximum loading of 8 μg/cm2. Because the PEM thin film can be coated on a wide range of surfaces, even those having very complex morphology, the PEM matrix could be a good potential host for curcumin, especially for drug-delivery patches or wound dressing applications.
Conclusions Multilayer thin films prepared from PDADMAC and PSS polyelectrolytes were used as a host matrix for the partitioning of curcumin. The extinction coefficient of curcumin in the PSS/ PDADMAC film is equal to 64 000 M-1 cm-1, and the partition coefficient of curcumin in the same film for a solvent of 80/20% v/v water/ethanol was calculated to be 2.07 105. The loading of curcumin was found to occur through a bulk process whereby the amount and dose of curcumin loaded into the film could be controlled by changing the number of deposited layers. The loading was independent of the top layer, and the solvent quality was found to control the partition mechanism and allow control over the loading of curcumin in the film. Overall, this study has shown that PDADMAC/PSS thin films are a promising matrix for incorporating curcumin and other hydrophobic drugs, which could be of interest in drug-delivery applications. Acknowledgment. We are grateful for the financial support provided by the Center for Innovative Nanoscience and Nanotechnology (CIN grant 45-1.2/2552), Chulalongkorn University, the Thailand Graduate Institute of Science and Technology (TG-55-09-51-034D) and the Thai Research Fund (TRF grant TRG5280034).
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