pubs.acs.org/Langmuir © 2010 American Chemical Society
Formation of Biocompatible Nanocapsules with Emulsion Core and Pegylated Shell by Polyelectrolyte Multilayer Adsorption K. Szczepanowicz,*,† H. J. Hoel,‡ L. Szyk-Warszynska,† E. Bielanska,† A. M. Bouzga,§ G. Gaudernack,‡ C. Simon,§ and P. Warszynski† † Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezpominajek 8, Krakow 30-239, Poland, ‡Institut for Cancer Research at the Norwegian Radium Hospital, Oslo University Hospital, Montebello, N-0310, Oslo, Norway, and §SINTEF Material and Chemistry, Forskningsveien 1, NO-0314 Oslo, Norway
Received May 21, 2010. Revised Manuscript Received June 23, 2010 The aim of this work was to develop a novel method of preparation of loaded nanosize capsules based on liquid core encapsulation by biocompatible polyelectrolyte (PE) multilayer adsorption, with or without pegylated outermost layer. Using AOT (docusate sodium salt) as emulsifier, we obtained cores, stabilized by an AOT/PLL (poly-L-lysine hydrobromide) surface complex. These positively charged cores were encapsulated by layer-by-layer adsorption of polyelectrolytes, biocompatible polyanion PGA (poly-L-glutamic acid sodium salt), and biocompatible polycation PLL. We used the saturation method for formation of consecutive layers, and we determined the optimal conditions concerning concentration of surfactant and polyelectrolytes to form stable shells. The average size of the obtained capsules was 60 nm. Pegylated external layer were prepared using PGA-g-PEG (PGA grafted by PEG poly(ethylene glycol)). The capsules were stable for at least a period of 3 months. These nanocapsules were biocompatible when tested for cytotoxicity in a cellular coculture assay and demonstrated no or very low nonspecific binding to peripheral blood mononuclear cells when tested by flow cytometry. In order to study drug effects on leukemia cells, β-carotene and vitamin A have been encapsulated as model drugs.
Introduction The development of modern nanocontainers is currently one of the main topics in pharmaceutical research. In the past years major progress in the synthesis and application of various types of microcontainers has been achieved.1-4 They can be obtained in the encapsulation is the process, in which colloidal particles or droplets are being coated by shells of various materials. Nanoencapsulation, i.e., the development of nanosize capsules, has the potential to improve the solubility of lipophilic, poorly watersoluble or even water-insoluble compounds and can protect molecules from the biological environment. Nanocapsules can be also used in specific drug delivery system as they can penetrate the cell membrane. The layer-by-layer (LbL) adsorption of polyelectrolytes (PE) is considered as a convenient method to obtain microcapsules’ shells on colloidal cores.5-10 Solid particles (polystyrene latex, silica, CaCO3) are most often used as cores for formation of capsules; then the solid core can be dissolved to leave the hollow shell, which can be then refilled with the desired composition.4-9 The main disadvantages of this method are *To whom correspondence should be addressed. (1) White, S. R.; Sottos, N. R.; Geubelle, P. H.; Moore, J. S.; Kessler, M. R.; Sriram, S. R.; Brown, E. N.; Viswanathan, S. Nature 2001, 409, 794–797. (2) Sukhorukov, G. B.; Fery, A.; Brumen, M.; Mohwald, H. Phys. Chem. Chem. Phys. 2004, 6, 4078–4089. (3) Guzey, D.; McClements, D. J. Adv. Colloid Interface Sci. 2006, 227, 128–130. (4) Landfester, K. Annu. Rev. Mater. Res. 2006, 36, 231–279. (5) Sukhorukov, G. B.; Donath, E.; Lichtenfeld, H.; Knippel, H.; Knippel, M.; Budde, A.; Moehwald, H. Colloids Surf., A 1998, 137, 253–266. (6) Borodina, T. N.; Rumsh, L. D.; Kunizhev, S. M.; Sukhorukov, G. B.; Vorozhtsov, G. N.; Fel’dman, B. M.; Markvicheva, E. A. Biomed. Khim. 2007, 53, 557–565. (7) Mauser, T.; Dejugnat, C.; M€ohwald, H.; Sukhorukov, G. B. Langmuir 2006, 20, 5888–5893. (8) Shchukin, D. G.; Sukhorukov, G. B.; M€ohwald, H. Angew. Chem., Int. Ed.. 2003, 42, 4472–4475. (9) Shchukin, D. G.; Shutava, T.; Shchukina, E.; Sukhorukov, G. B.; Lvov, Y. M. Chem. Mater. 2004, 16, 3446–3451. (10) Voigt, A.; Lichtenfeld, H.; Sukhorukov, G. B.; Zastrow, H.; Donath, E.; Baumler, H.; Mohwald, H. Ind. Eng. Chem. Res. 1999, 38, 4037–4043.
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remains of the destructed core trapped in the capsule and low efficiency of loading of the active substance into the hollow shells.11,12 Application of emulsions droplets as liquid cores gives a possibility to encapsulate oil-soluble active components with control of size and shell properties of the obtained capsules and opens perspectives for application in many fields such as cosmetic, medicine, pharmacy, and food industry.13-17 For example, capsules with liquid cores can be used in drug delivery systems or as microreactors.18-21 A commonly used polymer pair for formation of multilayer films consists of standard synthetic polyelectrolytes like poly(styrenesulfonate) (PSS), poly(allylamine hydrochloride) (PAH), and poly(diallyldimethylammonium chloride) (PDADMAC). Only a few groups have been working on nature-based polyelectrolytes.22-27 Immobilization of pegylated corona is a one of (11) Grigoriev, D. O.; Bukreeva, T.; Moehwald, H.; Shchukin, D. G. Langmuir 2008, 24, 999–1004. (12) Sukhorukov, G. B.; Fery, A.; Brumen, M.; M€ohwald, H. Phys. Chem. Chem. Phys. 2004, 6, 4078–4089. (13) Georgieva, R.; Moya, S.; Donath, E.; Baumler, H. Langmuir 2004, 20, 1895–1900. (14) Sukhorukov, G. B.; D€ahne, L.; Hartmann, J.; Donath, E.; Mohwald, H. Adv. Mater. 2000, 12, 112–115. (15) Caruso, F.; Fiedler, H.; Haage, K. Colloids Surf., A 2000, 169, 287–293. (16) Schuler, C.; Caruso, F. Biomacromolecules 2001, 2, 921–926. (17) Szczepanowicz, K.; Dronka-Gora, D.; Para, G.; Warszynski, P. J. Microencapsulation 2010, 27, 198–204. (18) Donath, E.; Moya, S.; Neu, B.; Sukhorukov, G. B.; Georgieva, R.; Voigt, A.; Baeumler, H.; Kiesewetter, H.; M€ohwald, H. Chem.;Eur. J. 2002, 8, 5481– 5485. (19) Radtchenko, I. L.; Giersig, M.; Sukhorukov, G. B. Langmuir 2002, 18, 8204–8208. (20) Shchukin, D. G.; Radtchenko, I. L.; Sukhorukov, G. B. J. Phys. Chem. B 2003, 107, 86–90. (21) Shchukin, D. G.; Sukhorukov, G. B.; M€ohwald, H. J. Phys. Chem. B 2004, 108, 19109–19113. (22) Berth, G.; Voigt, A.; Dautzenberg, H.; Donath, E.; M€ohwald, H. Biomacromolecules 2002, 3, 579–590. (23) R€ube, A.; Hause, G.; M€ader, K.; Kohlbrecher, J. J. Controlled Release 2005, 107, 244–252.
Published on Web 07/07/2010
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the available methods to prevent serum protein adsorption and nonspecific bindings to cells by the particles.28,29 A conventional method to immobilize PEG on polyelectrolytes microcapsules is the use of copolymers with PEG chains grafted to a polyelectrolyte backbone. Heuberger et al.29 used PLL-g-PEG, consisting of a polycationic PLL backbone whose side chain amino groups were partially grafted with PEG chains. The analogous route was proposed by Boulmedais et al.30 A negatively charged poly(Lglutamic acid) (PGA) backbone was partially grafted by PEG chains, leading to PGA-g-PEG. The grafting ratio, g, defined as the total number of monomers (lysine or glutamic acid) divided by the number of PEG side chains, was found to have a major influence on the stealth effect, with an optimum around g = 3.5 for PLL-g-PGA.31 In addition, the PEG side chains can be functionalized with bioligands while retaining resistance to unspecific protein adsorption.32-35 The results of our previous work17 have indicated that formation of stable interfacial complex of an ionic surfactant and an oppositely charged polyelectrolyte requires selection of surfactant, which provides a stable surface charge of oil/water interface, i.e., does not desorb from the interface on contact with polyelectrolyte solution. It was demonstrated that use of the negatively charged oil-soluble surfactant AOT (docusate sodium salt: FDA approved anionic oil-soluble surfactant) as emulsifier led to formation of octane emulsion droplets, which were stabilized by AOT/PDADMAC (poly(diallyldimethylammonium chloride)) surface complexes.17 That positively charged octane liquid cores, stabilized by AOT/PDADMAC complex, could be encapsulated by layer-by-layer adsorption of polyanion/polycation pairs. The aim of our present work was to extend that method of preparation of nanocapsules to produce nanocontainers loaded with model drugs like β-carotene and vitamin A. The method is based on the direct encapsulation of emulsion drops by multilayer adsorption, using biocompatible polyelectrolytes (PLL, PGA) with pegylated outermost layer. We also verified the toxicity of the resulting nanocapsules and the nonspecific binding to blood cells.
Experimental Section Materials. The polyelectrolytes used in our studies were polycations [poly(L-lysine hydrobromide), PLL (MW ∼ 15 00030 000), poly(fluorescein isothiocyanate allylamine hydrochloride), FITC-PAH (MW ∼ 70 000), poly(allylamine hydrochloride), PAH (MW ∼ 70 000)] and polyanion [poly(L-glutamic acid) sodium salt, PGA (MW ∼ 15 000-50 000)]. All polyelectrolytes [methoxypoly(ethylene glycol) amine (MW ∼ 5000), N-hydroxysulfosuccinimide (24) Ogawa, S.; Decker, E.; McClements, D. J. Agric. Food Chem. 2004, 52, 3595–3600. (25) Preetz, C.; R€ube, A.; Reiche, I.; Hause, G.; M€ader, K. Nanomedicine 2008, 4, 106–114. (26) Thiele, L.; Rothen-Rutishauser, B.; Jilek, S.; Wunderli-Allenspach, H.; Merkle, H. P.; Walter, E. J. Controlled Release 2001, 76, 59–71. (27) Tang, Z.; Wang, Y.; Podsiadlo, P.; Kotov, N. A. Adv. Mater. 2006, 18, 3203–3224. (28) Harris, J. M., Zalipsky, S., Eds.; ACS Symposium Series No. 680; American Chemical Society: Washington, DC, 1997. (29) Heuberger, R.; Sukhorukov, G.; V€or€os, J.; Textor, M.; M€ohwald, H. Adv. Funct. Mater. 2005, 15, 357. (30) Boulmedais, F.; Frisch, B.; Etienne, O.; Lavalle, P.; Picart, C.; Ogier, J.; Voegel, J. C.; Schaaf, P.; Egles, C. Biomaterials 2004, 25, 2003–2011. (31) Wattendorf, U.; Koch, M. C.; Walter, E.; V€or€os, J.; Textor, M.; Merkle, H. P. Biointerphases 2006, 1, 123–133. (32) Tosatti, S.; De Paul, S. M.; Askendal, A.; VandeVondele, S.; Hubbell, J. A.; Tengvall, P.; Textor, M. Biomaterials 2003, 24, 4949–4958. (33) Huang, N. P.; V€or€os, J.; De Paul, S. M.; Textor, M.; Spencer, N. D. Langmuir 2002, 18, 220–230. (34) Van de Vondele, S.; V€or€os, J.; Hubbell, J. A. Biotechnol. Bioeng. 2003, 82, 784–790. (35) M€uller, M.; V€or€os, J.; Csucs, G.; Walter, E.; Danuser, G.; Merkle, H. P.; Spencer, N. D.; Textor, M. J. Biomed. Mater. Res., Part A 2003, 66A, 55–61.
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Article Table 1. Zeta Potential of Nanocapsules Measured in 0.015 M NaCl nanocapsules
zeta potential [mV]
AOT(PLL/PGA)2,5 AOT(PLL/PGA)2,5PGA-g-PEG AOT(PLL/PGA)3,5 AOT(PLL/PGA)3,5 PGA-g-PEG
43 ( 4 -4 ( 6 39 ( 5 -3 ( 4
sodium salt, NHS (g98.5%), N-(3-(dimethylamino)propyl)-N0 ethylcarbodiimide, EDC (g97%), docusate sodium salt, AOT (g99%), 3-(2-benzothiazolyl)-7-(diethylamino)coumarin, Coumarine-6 (98%), sodium chloride, β-carotene, vitamin A] were obtained from Sigma-Aldrich. Chloroform cz.d.a. was purchased from POCH Gliwice. All materials were used without further purification. The distilled water used in all experiments was obtained with the three-stage Millipore Direct-Q 3UV purification system. Synthesis of PGA-g-PEG. PGA-g-PEG was synthesized according to the procedure described by Boulmedais et al. 0.041 g (0.272 mmol in acid function) of PGA, 0.43 g (0.085 mmol of amine function, in order to obtain a grafting ratio of 32%) of MePEGNH2, and 0.01 g (0.05 mmol) of NHS were dissolved in 10 mL of water. EDC (0.068 g) (0.44 mmol) was dissolved in the mixture with stirring. The reaction was allowed to proceed for 6 h at room temperature. After filtration, the reaction mixture was dialyzed 4 times (cutoff at MW 12 400) for 24 h with deionized water (2 L). The achieved coupling rate of ∼31% (31 PEG for 100 monomer of glutamic acid), corresponding to a grafing ratio of g = 3.2 as determined by 1H NMR. The zeta potential of PGA-g-PEG was measured in 0.015 M NaCl. Capsules Preparation. Capsules were prepared using a modified method proposed by Szczepanowicz et al.17 The oil phase for capsules preparation was prepared by dissolution of 360 g/dm3 AOT in chloroform. Polyelectrolytes were dissolved in NaCl solutions of ionic strength (0.015 M) to obtain PE solution of concentration 1 g/dm3, without any pH adjustment. Nanocapsules were formed by addition of AOT/chloroform to polycation (PLL) solution during mixing with a magnetic stirrer at 300 rpm. The optimal ratio of surfactant (AOT) to polycation concentrations was determined by measuring zeta potential of emulsion drops and examining their stability. Stable emulsion was obtained when zeta potential of drops with adsorbed polyelectrolyte layer was close to zeta potential of the same polyelectrolyte in solution. That way we minimized the amount of free polyelectrolyte in the suspension. It is also worth noting that in our method of preparation of emulsion cores we avoid the presence of surfactants in the aqueous phase, which is usually toxic to cells. To encapsulate model drugs, β-carotene or vitamin A was dissolved in chloroform (0.1 mg/mL) prior to emulsification with AOT. After adsorption of the first layer of polycation, the consecutive layers of polyelectrolytes were formed by layer-bylayer technique, using the saturation method.3,5,36 Therefore, the multilayer shells were constructed by subsequent adsorption of polyelectrolytes from their solutions without the intermediate rinsing step. Suspension of capsules was added to the polyelectrolyte solution while mixing with magnetic stirrer at 300 rpm. Volumes of polyelectrolyte solution used to form each layer were chosen empirically by analyzing the results of simultaneous zeta potential measurements. It was found optimal, when the zeta potential reached a value, close to the zeta potential of the same polyelectrolyte in solution. To create pegylated shell, PLL-terminated nanocapsules with five and seven polyelectrolytes layers were coated with layer of PGA-g-PEG using the same procedure as described above, by adding nanocapsules (PLL terminated) into filtered PGA-g-PEG polymer solution. For preparation fluorescently labeled nanocapsules FITC-PAH was used instead one PLL layer. (36) Volodkin, D. V.; Larionova, N. I.; Sukhorukov, G. B. Biomacromolecules 2004, 5, 1962–1972.
DOI: 10.1021/la102061s
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Particle Size Analysis. The size distribution (hydrodynamic diameter) of capsules was determined by DLS (dynamic light scattering) using a Zetasizer Nano Series from Malvern Instruments with the detection angle of 173° in optically homogeneous square polystyrene cells. Each value was obtained as average from three runs with least 10 measurements. All measurements were performed at 25 °C. Zeta Potential Measurements. The zeta potential of capsules was measured by the microelectrophoretic method using Malvern Zetasizer Nano ZS apparatus. Each value was obtained as an average from three subsequent runs of the instrument with at least 20 measurements. The zeta potential of capsules as well as of polyelectrolytes in solution was measured in 0.015 M NaCl. Fluorescence Microscopy and Spectroscopy. To visualized nanocapsules, oil-soluble fluorescent dye Coumarine-6 was dissolved in chloroform (0.1 mg/mL) prior to emulsification with AOT. Images of so-prepared suspensions were taken with a Carl Zeiss Observer D1 microscope with TIRF illumination. Fluorescent emission spectra were also measured to confirm encapsulation of the model drug vitamin A. Spectra was obtained using Spectrofluoremeter Fluorolog-3 from Jobin Yvon Inc. 1 H NMR Spectroscopy. 1H NMR spectroscopy was applied to characterize PEG grafting ration of synthesized PGA-g-PEG. Spectra were obtained using a Bruker AVANCE 400 MHz 400.13 MHz for 1H, Probehead: BBFO Plus with Z-gradients (5 mm PABBO-BB-1H/D Z-GRD). The volume of 0.7 mL of the PGA-gPEG in D2O was used in the NMR tube. SEM (Scanning Electron Microscopy). Microscopic SEM (scanning electron microscope) observations of nanocapsules were performed with a JEOL JSM-7500F, field emission scanning electron microscope, at an operation voltage of 15 keV. Samples were prepared by immersing coppers cylinder in nanocapsules suspension and drying overnight. UV-vis. UV-vis spectrometry was applied to confirm encapsulation of model drugs. UV/vis absorption spectra of the capsules were acquired by using an Analytik Jena AG-SPECORD 40 spectrophotometer. Stability Studies. To assess the stability of nanocapsules with the PGA-g-PEG outermost layer, freshly prepared nanocapsules were stored in 0.015 M NaCl solution at room temperature for up to 90 days. Size distribution (hydrodynamic diameter) and zeta potential of pegylated nanocapsules were measured after 1, 7, 14, 30, and 90 days as described above. Cytotoxicity Tests. The cytotoxicity of the samples of nanocapsules was tested in a proliferation assay. In this assay the samples are cultured in RPMI 1640 (PAA Laboratories GmbH, Pasching, Austria) and Garamycin (Schering-Plough Labo, Heistop-den-Berg, Belgium) with B-LCL cells (a lymphoblastoid cell line capable of indefinite growth) and 3.7 104 Bq [3 H]thymidine (American Radiolabeled Chemicals, Inc., St. Louis, MO). Cells and nanocapsules are cultured in triplicate wells in round bottomed plates (Costar, Corning Inc., Corning, NY). After ∼20 h the cultures are harvested onto UniFilter plates (PerkinElmer Inc., Waltham, MA), dried at 45 °C for 1.5 h before 25 μL of Microscint O (PerkinElmer Inc.) is added to the filters. The amount of thymidine incorporated into the DNA of dividing cells can then be measured in a microplate scintillation counter (PerkinElmer Inc.). Results are calculated from mean of triplicates and expressed as percent 3H incorporation relative to control cultures without nanocapsules. For the cytotoxicity tests AOT(PLL/PGA)3,5PGA-g-PEG nanocapsules were chosen, and the results of tests were compared with ones for the emulsion drops stabilized by AOT/PAH surface complexes. Tests of Unspecific Binding. To evaluate the unspecific binding of the fluorescently labeled AOT(PLL/PGA)3,5, PGA-g-PEG nanocapsules (with one FITC-PAH layer instead PLL) were incubated with peripheral blood mononuclear cells (PBMC) isolated from the blood of healthy donors. The nanocapsules sample concentrations used were those found to be 12594 DOI: 10.1021/la102061s
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Figure 1. Typical size distribution of emulsion drops: chloroform/ AOT-PLL/water measured by DLS. In case of using (a) 0.2 g/dm3 and (b) 0.5 g/dm3 aqueous PLL solution. nontoxic to cells in the proliferation assays. PBMCs were incubated in CellGro DC medium (CellGenix, Freiburg, Germany) with Garamycin (Schering-Plough Labo) in 48 well plates (Costar, Corning Inc., Corning, NY), and nanocapsules samples diluted in the same medium were added to the cells. The cells were incubated with nanocapsules at 37 °C for 3 h before they were washed once in medium and resuspended in phosphate buffered saline (PBS) for analysis of fluorescence on a BD LSR II flow cytometer (BD, NJ). The results were analyzed in FlowJo Software (Tree Star, Inc., Ashland, OR).
Results and Discussion For preparation of the suspension of nanocapsules, 0.1 mL of AOT in chloroform solution was added to 200 mL of aqueous PLL solution (c = 0.1 g/dm3) during continuous mixing. The concentration of obtained emulsion was 0.05 vol %. Chloroform was evaporated from suspensions of nanocapsules after preparation. The average drop size measured by DLS was around 60 nm (Figure 1a) with polydispersity index (PDI)
