pubs.acs.org/Langmuir © 2009 American Chemical Society
Surface-Active Monomer as a Stabilizer for Polyurea Nanocapsules Synthesized via Interfacial Polyaddition in Inverse Miniemulsion Eva-Maria Rosenbauer,†,‡ Katharina Landfester,†,‡ and Anna Musyanovych*,†,‡ †
Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany, and ‡Institute of Organic Chemistry III - Macromolecular Chemistry and Organic Materials University of Ulm, Albert-EinsteinAllee 11, 89081 Ulm, Germany Received May 13, 2009. Revised Manuscript Received June 25, 2009 A surface-active monomer, polyisobutylene-succinimide pentamine (Lubrizol U), was used as a stabilizer for synthesizing polyurea nanocapsules with aqueous core via polyaddition at inverse miniemulsion droplet interface. Because of the presence of amine groups in the Lubrizol molecule, it is covalently incorporated into the polymeric interfacial layer after reaction, resulting in more compact (less permeable) capsule shell. The influence of the stabilizer and the monomer concentration on the shell thickness, colloidal stability, average capsule size, and capsule size polydispersity were examined in detail. Different materials, such as a water-soluble fluorescent dye and aqueous dispersion of magnetite nanoparticles with 10 nm in size, were used as inner phase of the polyurea capsules. The encapsulation efficiency was studied using fluorescein as a marker. As an example for biomedical application, the fluorescein-containing capsules were utilized in cell uptake experiments and visualized using fluorescence microscopy.
1. Introduction The preparation of polymeric nanocapsules has attracted a widespread interest in the past decades due to their potential applications in numerous fields, including sensors, vesicles for enzymes and chemicals, controlled drug delivery systems, etc.1-3 Particularly stable polymeric nanocapsules with an aqueous core are of high importance for the protection, storage, and delivery of hydrophilic organic/inorganic compounds. Generally, there a two major approaches to synthesize polymeric nanocapsules, which can be differentiated by the presence or absence of a sacrificial core template. In the first method the core particles, usually of mineral or organic silica origin, are used as templates. The particles can then be modified with a polymeric shell or different polymeric layers by adsorption of a preformed polymer or by the direct polymerization onto the core surface. Afterward, the template cores are removed under physical or chemical conditions by dissolution or calcination process resulting in the hollow nanospheres, which can be subsequently loaded with a preferred target. Feldheim et al. have used gold nanoparticles as templates for the synthesis of polypyrrole and poly(N-methylpyrrole) shells.4 After etching the gold, the hollow polymer capsules with a shell thickness governed by the polymerization time were obtained. The synthesis of fluorescent thermosensitive poly(Nisopropylacrylamide) (PNIPAM) nanocapsules with temperature-tunable size and shell permeability was reported by Gao et al.5 First, the cross-linked PNIPAM shell was prepared by precipitation polymerization in the presence of the isothiocyanate fluorescein (FITC)-trapped silica particles serving as seeds. Then, the SiO2 core was etched by hydrofluoric acid (HF), and the *Corresponding author. E-mail:
[email protected]. (1) Sukhorukov, G.; Fery, A.; M€ohwald, H. Prog. Polym. Sci. 2005, 30(8-9), 885–897. (2) Yang, S.; Zhang, Y.; Yuan, G.; Zhang, X.; Xu, J. Macromolecules 2004, 37 (26), 10059–10062. (3) Chu, L. Y.; Yamaguchi, T.; Nakao, S. Adv. Mater. 2002, 14(5), 386–389. (4) Marinakos, S. M.; Novak, J. P.; Brousseau, L. C.III; House, A. B.; Edeki, E. M.; Feldhaus, J. C.; Feldheim, D. L. J. Am. Chem. Soc. 1999, 121(37), 8518–8522. (5) Gao, H.; Yang, W.; Min, K.; Zha, L.; Wang, C.; Fu, S. Polymer 2005, 46 (4 spec. iss.), 1087-1093.
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entrapped FITC molecules remained within the capsules cavity. The formation of a polymeric shell by adsorption on the template core can be also achieved using the layer-by-layer technique, which is based on an alternate adsorption of oppositely charged polyelectrolytes (or nanoparticles) onto the fluid droplet surface6 or onto the surface of a sacrificial core. The thickness of the capsule shell can be adjusted by the number of layer-by-layer deposition cycles. The size of the capsule depends on the colloidal template size. Capsules with diameter varied from 70 nm to 10 μm can be obtained.6-12 Direct shell polymerization around the particle core was described recently involving dispersion13-15 or living polymerization mechanisms (e.g., atom transfer radical polymerization or anionic polymerization).16,17 Later, the core was eliminated by chemical etching with HF or THF.18-24 (6) Li, J. B.; Mohwald, H.; An, Z. H.; Lu, G. Soft Matter 2005, 1(4), 259–264. (7) Caruso, F.; Spasova, M.; Salgueirino-Maceira, V.; Liz-Marzan, L. M. Adv. Mater. 2001, 13(14), 1090–1094. (8) Caruso, F. Adv. Mater. 2001, 13(1), 11–22. (9) Caruso, F.; Caruso, R. A.; M€ohwald, H. Chem. Mater. 1999, 11(11), 3309– 3314. (10) Caruso, F.; Caruso, R. A.; M€ohwald, H. Science 1998, 282(5391), 1111– 1114. (11) Park, M. K.; Xia, C.; Advincula, R. C.; Sch€utz, P.; Caruso, F. Langmuir 2001, 17(24), 7670–7674. (12) Wang, K. W.; He, Q.; Yan, X. H.; Cui, Y.; Qi, W.; Duan, L.; Li, J. B. J. Mater. Chem. 2007, 17, 4018–4021. (13) Xu, X.; Asher, S. A. J. Am. Chem. Soc. 2004, 126(25), 7940–7945. (14) Bourgeat-Lami, E.; Lang, J. J. Colloid Interface Sci. 1998, 197(2), 293–308. (15) Bourgeat-Lami, E.; Lang, J. J. Colloid Interface Sci. 1999, 210(2), 281–289. (16) Ali, M. M.; St€over, H. D. H. Macromolecules 2003, 36(6), 1793–1801. (17) Ali, M. M.; St€over, H. D. H. J. Polym. Sci., Part A: Polym. Chem. 2006, 44 (1), 156–171. (18) Perruchot, C.; Khan, M. A.; Kamitsi, A.; Armes, S. P.; Von Werne, T.; Patten, T. E. Langmuir 2001, 17(15), 4479–4481. (19) Von Werne, T.; Patten, T. E. J. Am. Chem. Soc. 2001, 123(31), 7497–7505. (20) Fleming, M. S.; Mandal, T. K.; Walt, D. R. Chem. Mater. 2001, 13(6), 2210–2216. (21) Mori, H.; Seng, D. C.; Zhang, M.; M€uller, A. H. E. Langmuir 2002, 18(9), 3682–3693. (22) Kamata, K.; Lu, Y.; Xia, Y. J. Am. Chem. Soc. 2003, 125(9), 2384–2385. (23) Mandal, T. K.; Fleming, M. S.; Walt, D. R. Chem. Mater. 2000, 12(11), 3481–3487. (24) Zhou, Q.; Wang, S.; Fan, X.; Advincula, R.; Mays, J. Langmuir 2002, 18(8), 3324–3331.
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Preparing polymeric nanocapsules without any sacrificial core is more beneficial, since those capsules can be synthesized in fewer steps and the target material can be placed inside the “core” already in the beginning of the capsule process formation. There are several techniques that are commonly used to obtain capsules directly. For example, by the “ouzo effect”,25,26 an induced phase separation within emulsion or miniemulsion droplets,27,28 the selfassembly of block copolymers,29 cross-linking of polymerizable liposomes,30 templating of vesicles,31,32 via interfacial polymer deposition under solvent displacement,33-35 or by interfacial cross-linking reactions.36,37 A highly promising way to create polymeric nanocapsules in a controlled manner is via the miniemulsion process where monodisperse and stable droplets in the size range from 50 to 500 nm can be formed. The size of the droplets mainly depends on the type and effective amount of the surfactant used. The choices of monomers/ polymers and chemical reactions that can be utilized to form the nanocapsules with a hydrophilic or hydrophobic core are almost unlimited. Therefore, this technique gives the opportunity to produce capsules with desired properties for a broad range of applications. As an example, poly(methyl methacrylate) nanocapsules with an aqueous core containing antiseptic agent were obtained by controlled nanoprecipitation of a preformed polymer onto the droplet’s surface.38 Van Zyl et al.39 reported the synthesis of nanocapsules with a liquid core and molar mass-controlled polystyrene shell by an in situ miniemulsion polymerization reaction performed in the presence of a RAFT (reversible addition-fragmentation chain transfer) agent. By carrying out the anionic polymerization at the interface of stable water-in-oil droplets, poly(butyl cyanoacrylate) nanocapsules containing DNA molecules were prepared.40,41 Polyurethane nanocapsules with a hydrophobic core were obtained via the interfacial polyaddition in the direct miniemulsions.42,43 Previously, polyurethane and polyurea nanocapsules with an aqueous core were successfully formulated performing the interfacial polyaddition/polycondensation reactions in the inverse miniemulsion system.44,45 Nanocapsules produced in such a way could be easily loaded with (25) Ganachaud, F.; Katz, J. L. ChemPhysChem 2005, 6(2), 209–216. (26) Vitale, S. A.; Katz, J. L. Langmuir 2003, 19(10), 4105–4110. (27) Dowding, P. J.; Atkin, R.; Vincent, B.; Bouillot, P. Langmuir 2004, 20(26), 11374–11379. (28) Tiarks, F.; Landfester, K.; Antonietti, M. Langmuir 2001, 17(3), 908–918. (29) Wong, M. S.; Cha, J. N.; Choi, K. S.; Deming, T. J.; Stucky, G. D. Nano Lett. 2002, 2(6), 583–587. (30) Lestage, D. J.; Urban, M. W. Langmuir 2005, 21(10), 4266–4267. (31) Hotz, J.; Meier, W. Adv. Mater. 1998, 10(16), 1387–1390. (32) Hotz, J.; Meier, W. Langmuir 1998, 14(5), 1031–1036. (33) Fessi, H.; Puisieux, F.; Devissaguet, J. P.; Ammoury, N.; Benita, S. Int. J. Pharm. 1989, 55(R1-R4), 1590. (34) Crespy, D.; Landfester, K. Macromol. Chem. Phys. 2007, 208(5), 457–466. (35) Alvarez-Roman, R.; Barre, G.; Guy, R. H.; Fessi, H., Eur. J. Pharm. Biopharm. 2001, 52(2), 191–195. (36) Soto-Portas, M. L.; Argillier, J. F.; Mechin, F.; Zydowicz, N. Polym. Int. 2003, 52(4), 522–527. (37) Sun, Q.; Deng, Y. J. Am. Chem. Soc. 2005, 127(23), 8274–8275. (38) Paiphansiri, U.; Tangboriboonrat, P.; Landfester, K. Macromol. Biosci. 2006, 6(1), 33–40. (39) Van Zyl, A. J. P.; Bosch, R. F. P.; McLeary, J. B.; Sanderson, R. D.; Klumperman, B. Polymer 2005, 46(11), 3607–3615. (40) Musyanovych, A.; Landfester, K. Colloid Polym. Sci. 2008, in press. (41) Lambert, G.; Fattal, E.; Pinto-Alphandary, H.; Gulik, A.; Couvreur, P. Pharm. Res. 2000, 17(6), 707–714. (42) Torini, L.; Argillier, J. F.; Zydowicz, N. Macromolecules 2005, 38(8), 3225– 3236. (43) Johnsen, H.; Schmid, R. B. J. Microencapsulation 2007, 24(8), 731–742. (44) Landfester, K.; Tiarks, F.; Hentze, H. P.; Antonietti, M. Macromol. Chem. Phys. 2000, 201(1), 1–5. (45) Crespy, D.; Stark, M.; Hoffmann-Richter, C.; Ziener, U.; Landfester, K. Macromolecules 2007, 40(9), 3122–3135. (46) Jagielski, N.; Sharma, S.; Hombach, V.; Mail€ander, V.; Rasche, V.; Landfester, K. Macromol. Chem. Phys. 2007, 208(19-20), 2229–2241.
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Figure 1. Lubrizol U (polyisobutylene-succinimide pentamine). Length of the polyisobutylene chain, n = 1-10.
contrast agents, like Magnevist and Gadovist for applications in magnetic resonance imaging (MRI).46 For an efficient stabilization of the aqueous droplets in a hydrophobic continuous phase, only a limited number of surfactants can be applied. Usually different types of sorbitan esters, known also as Span, or specially synthesized block copolymers are used. With the aim to increase the capsules stability and encapsulation efficiency of low molecular weight compounds, in the current paper we used polyisobutylene-succinimide pentamine (Lubrizol U, for structure see Figure 1). Because of the presence of amine groups, this molecule acts in polyaddition reaction as a monomer as well as a stabilizing agent at the same time and can therefore be considered as “surfmer”. After polymerization the surfmer is expected to be covalently bounded into the polymeric interfacial layer, leading to the improved properties of the capsules, e.g., more compact and impermeable shell. The influences of the surfactant and monomer concentration on the shell thickness, colloidal stability, average capsule size, and capsule size polydispersity were examined in detail. Furthermore, the conversion of monomer into polymer was studied by FTIR, and the incorporation efficiency of Lubrizol was estimated from the GPC results. To show the versatility in application of the obtained capsules, different materials, such as a water-soluble fluorescent dye and aqueous dispersion of magnetite nanoparticles with 10 nm in size, were used as inner phase of the polyurea capsules. The encapsulation efficiency was studied using fluorescein as a marker.
2. Experimental Section 2.1. Materials and Methods. 1,6-Diaminohexane (HMDA, Fluka), toluene 2,4-diisocyanate (TDI, 98%, Aldrich), and cyclohexane (HPLC-grade) were used without further purification. Sodium dodecyl sulfate (SDS, 99% Merck) and Lubrizol U (polyisobutylene-succinimide pentamine, Mw = 384-875 g mol-1, determined from GPC, HLB < 7, containing 50:50 w/w % mineral oil as a diluent, Lubrizol, France) were used as surfactants. Fluorescein (free acid, Riedel-de H€aen) was utilized as a hydrophilic fluorescent marker. Demineralized water and phosphate saline buffer (PBS, 0.15 M, pH 7.4) were used as an aqueous phase throughout the experiments. 2.2. Preparation of Polyurea Capsules. For the synthesis of polyurea capsules, a certain amount of HMDA (for details see Table 1) and 0.750 g of PBS buffer, which form the hydrophilic liquid cores, were homogenized for 3-5 min at room temperature. This mixture was added to 6 g of cyclohexane containing a defined amount of Lubrizol U. After pre-emulsification at room temperature for 1 h with a magnetic stirrer (in order to obtain an effective macroemulsion), the miniemulsion was prepared by ultrasonicating the mixture for 180 s at 70% amplitude (Branson sonifier W450 Digital, tip size 6.5 mm) under ice cooling for preventing evaporation of cyclohexane. Then a defined amount of TDI (for details see Table 1) dissolved in 4 g of cyclohexane was dropwisely added to the miniemulsion. The reaction was carried out for 3 h at 25 °C. The redispersion of the nanocapsules in the aqueous phase was performed by mixing DOI: 10.1021/la9017097
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Rosenbauer et al. Table 1. Reaction Composition and Characterization of Synthesized Polyurea Capsulesa average diameter, nm b
HMDA/TDI, molar ratio
HMDA, mol (mg) -4
TDI, mol (mg) -4
DLS (in cyclohexane)
DLS (in aqueous phase)
TEM (in cyclohexane)
2.6 10 -c 1:0.4 6.5 10 (1:0.39) (75) (28) 6.5 10-4 255 246 235 1:1 6.5 10-4 (1:0.97) (75) (112) -4 -4 9.8 10 280 242 224 1:1.5 6.5 10 (1:1.46) (75) (170) 1.3 10-3 440 350 268 1:2 6.5 10-4 (1:1.94) (75) (225) -4 -4 c 6.5 10 365 -c 1.5:1 9.8 10 (1.5:0.98) (112) (112) 6.5 10-4 -c -c -c 2:1 1.3 10-3 (2:0.98) (150) (112) a Each sample contains 0.75 g of PBS buffer as an aqueous phase and 10 g of cyclohexane with 25 mg of dissolved Lubrizol U as continuous phase. b The values in parentheses correspond to the HMDA þ Lubrizol U/TDI molar ratio. c The measurements were not possible due to the presence of broken capsules or their strong aggregation.
1.0 g of the synthesized nanocapsule cyclohexane dispersion with an aqueous solution of SDS (0.02 mol L-1) for 5 min. The resulting mixture was subjected to the ultrasonication for 60 s at 70% amplitude, 10 s pulse, 5 s pause (Branson sonifier W450 Digital, tip size 6.5 mm) under ice cooling, and the cyclohexane was evaporated afterward by heating the mixture up to 70 °C and stirring (400 rpm) for 30 min until ∼1.0 g of the suspension was removed. Polyurea capsules containing the fluorescent marker were prepared by adding 7.5 10-5 mol L-1 of fluorescein dye dissolved in PBS (0.15 M, pH 7.4) into the aqueous phase prior to pre-emulsification of both phases. The presence of ions in PBS converts free acid form of fluorescein into the salt form, therefore improving the water solubility of fluorescein. The encapsulation of magnetite was performed as followed: 75 mg of HMDA was mixed with 750 mg of an aqueous 4 wt % magnetite dispersion prepared according to ref 47. The homogenized solution was added dropwise to an organic phase of 6 g of cyclohexane and 50 mg of Lubrizol U. The resulting mixture was miniemulsified for 180 s at 70% amplitude (Branson sonifier W450 Digital, tip size 6.5 mm) under ice cooling after preemulsification for 1 h with a mechanical stirrer. Directly after ultrasonication, a solution containing 170 mg of TDI and 4 g of cyclohexane was added to the reaction mixture, and the reaction was carried out for 4 h at 25 °C under gentle stirring. 2.3. Characterization of Samples. The average size and size distribution of the final polymer capsules were determined by dynamic light scattering (DLS) using a Zeta Nanosizer (Malvern Instruments), equipped with a detector to measure the intensity of the scattered light at 173° to the incident beam. Transmission electron microscopy (TEM) (Philips EM400) was used to study the capsule morphology, the mean diameter, and the shell thickness of polymer capsules. An average of five TEM images of each miniemulsion sample, which contain not less than 100 nanocapsules, were analyzed, and an average capsule size and shell thickness were determined. The interfacial tension between demineralized water and the miniemulsion was determined using a commercial spinning drop tensiometer SVT 20N (DataPhysics, Germany). The obtained value was compared with the values of interfacial tension of cyclohexane/water and cyclohexane plus Lubrizol U/water in order to get the information about the coverage of the droplets with Lubrizol U. The working principle of the spinning drop tensiometer is based on the theory derived by Vonnegut.48 A glass capillary is filled with two fluids of interest, whereas the fluid with the lower density being in a minority, e.g., water (∼1.2 g) and miniemulsion (∼0.012 g) (see Figure 2). Then, the equipment tube was placed horizontally and equilibrated at (47) Ramı´ rez, L. P.; Antonietti, M.; Landfester, K. Macromol. Chem. Phys. 2006, 207(2), 160–165. (48) Vonnegut, B. Rev. Sci. Instrum. 1942, 13(1), 6–9.
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c
c
Figure 2. Schematic presentation of spinning drop experimental setup.
20 °C for several minutes under rotation at 6000-8000 rpm until “macroscopic” droplets at the axis of rotation were observed. At equilibrium, the shape of the droplet is a balance between interfacial (∼γ/a) and centrifugal (∼ΔFω2a2) stresses, where ΔF is the density difference between the phases. The interfacial tension of the fluid can be obtained using the Vonnegut formula: γ ¼
ðF1 -F2 Þω2 a3 4
In our experiment the L/a ratio of the droplet exceeds 4, stating that the droplet form is nearly cylindrical.48-50 FT-IR measurements were performed to follow the kinetics of the polyaddition. The samples were prepared using KBr pellets, and the measurements were carried out on a FT-IR 113v Bruker spectrophotometer equipped with a DTGS detector using 100 signal-averaged scans at a resolution of 2 cm-1. The solid used for the IR measurements was obtained by freeze-drying the samples from the cyclohexane or aqueous phases. The residual TDI concentration and the amount of nonreacted surfactant Lubrizol U were evaluated from GPC data. The samples were dissolved in cyclohexane, and the measurements were performed using an apparatus consisting of the Spectra System P2000 pump, an autosampler Agilent 1100, and two detectors, the Shodex differential refractometer RI-71 and the Knauer UV variable wavelength monitor detector. Separation was performed at a flow rate of 1 mL min-1 using two mixed-bed linear M columns (PSS, Mainz, Germany) with a particle size of 5 μm. The amount of TDI and Lubrizol was calculated by integrating the area under the GPC curve. The calibration curve obtained with a known amount of TDI (or Lubrizol U) was used to calculate the residual amounts of TDI and Lubrizol U in the experimental samples. The uptake studies into HeLa cells were studied after 24 h. For the incubation, HeLa cells were seeded at a density of 50 000 cells (49) Joseph, D. D.; Arney, M. S.; Gillberg, G.; Hu, H.; Hultman, D.; Verdier, C.; Vinagre, T. M. J. Rheol. 1992, 36(4), 621–662. (50) Princen, H. M.; Zia, I. Y. Z.; Mason, S. G. J. Colloid Interface Sci. 1967, 23 (1), 99–107.
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Rosenbauer et al. per cm2 on the first day. On the second day, fluorescein-containing capsules were added at a concentration of 75 μg mL-1 to the media. The intracellular localization of the capsules was confirmed by confocal laser scanning microscopy (CLSM, Fluoview on an IX71 equipped with two lasers, 488 and 543 nm, and a 60 oil lens, Olympus). The detailed procedure of the sample preparation is given in ref 51. The concentration of encapsulated fluorescein was measured in the supernatant obtained after centrifugation of the capsules redispersed in water. The fluorescent spectra were performed on a FluoroMax-3 spectrofluorometer (HORIBA Jobin Yvon, Inc.). The excitation wavelength was 490 nm, and the emission was measured at a wavelength of 520 nm. The calibration curve was obtained from the fluorescent intensities of defined fluorescein concentrations dissolved in SDS aqueous solution (0.02 mol L-1, pH 7.0). The total content of the fluorescein inside the capsules was calculated from the difference in fluorescent intensities between the calibration curve and the measured samples. The thermal properties of the capsule shell and the amount of encapsulated magnetite were determined from the thermogravimetric analysis (TGA) performed on a Perkin-Elmer TGA apparatus under a nitrogen atmosphere and heating rate of 10 °C min-1. Freeze-dried samples were used for the measurements. In the case of magnetite-containing capsules, the nonencapsulated magnetite was separated by gradient centrifugation, and the residual pellet of capsules was used for TGA measurements.
3. Results and Discussion A series of nanocapsules consisting of aqueous core and polyurea shell were prepared by an interfacial polyaddition reaction at the water-in-oil droplets interface,45 as shown in Figure 3. In the beginning of the formulation process, HMDA and fluorescent marker were mixed together in PBS buffer (0.15 M, pH 7.4), then added into the solution containing cyclohexane with different amounts of the Lubrizol U, and miniemulsified by ultrasonication, resulting in the narrow sized droplets in a size range between 100 and 500 nm. The salts presented in buffer serve as a costabilizer and prevents the Ostwald ripening of the droplets. Because of the hydrophilic character of HMDA, it is dissolved inside the droplets which are stabilized by the surfactant Lubrizol U. Afterward, a solution of the hydrophobic TDI in cyclohexane is dropwisely added to the miniemulsion. Because of the high reactivity of the amino groups in HMDA compared to the hydroxyl groups in water, the hydrophilic groups of the diisocyanate react more rapidly with the HMDA than with water, especially at room temperature.42 Moreover, the presence of the alkyl chain between two amino groups in HMDA determines that this molecule is amphiphilic and preferably stays at the droplet interface, and therefore, it can be assumed that the isocyanate groups can reach amino groups faster compared to the water hydroxyls concentrated inside the droplet. The polymer is formed simultaneously via polyaddition reaction and precipitates at the interfacial layer of the droplets. After synthesis in cyclohexane, the polyurea capsules were transferred into an aqueous solution of SDS. Lubrizol U is an efficient surfactant for a water-in-oil system, whereas SDS is required for the stabilization of nanocapsules in the aqueous medium in order to be suitable for biomedical applications. Although SDS is known to be not biocompatible, in the present work it is used as a model hydrophilic surfactant. Moreover, on the basis of the (51) Musyanovych, A.; Schmitz-Wienke, J.; Mail€ander, V.; Walther, P.; Landfester, K., Macromol. Biosci. 2008, 8(2), 127–139. (52) Lorenz, M. R.; Holzapfel, V.; Musyanovych, A.; Nothelfer, K.; Walther, P.; Frank, H.; Landfester, K.; Schrezenmeier, H.; Mail€ander, V. Biomaterials 2006, 27(14), 2820–2828.
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Figure 3. Synthesis of polyurea nanocapsules in the inverse (water-in-oil) miniemulsion system.
results of our previous cell uptake experiments,51,52 we did not observe any toxic effects of SDS on the cell vitality (within the experimental concentrations, i.e., 0.02 mol L-1). 3.1. Variation of HMDA to TDI Ratio. In order to study the influence of HMDA and TDI amounts on the average size and shell thickness of the capsules, the formulation process was carried out with different molar ratios of these reagents keeping the other parameters constant. The reaction composition and characterization of the final polyurea capsules are summarized in Table 1. The morphological studies of the obtained nanoparticles were performed by TEM. Some selected TEM images are presented in Figure 4. On the basis of the DLS and TEM results, it can be concluded that stable and well-defined polymeric capsules within a size range between 255 and 440 nm were obtained when the amount of HMDA was equal or less in molar ratio compared to the TDI concentration. In the case of using an excess of TDI, after the consumption of the existing amine groups (of HMDA and also of the surfactant, see below), the isocyanate groups can react with a water leading to the primary amine groups formation.53 These amine groups could then react with the isocyanate group again (if any still present) and be built into the polymeric chain. For the same systems, the polydispersity index (PDI) was in the range of 0.1-0.2, indicating a narrow size distribution. It is interesting to note that, according to the principle of miniemulsion technique, the capsule size should not be affected by the amount of added TDI. In our case the increase in the average diameter with the decrease in the HMDA/TDI molar ratio was clearly observed. This could be assigned to the reaction of TDI with Lubrizol U molecules dissolved in the continuous phase. In order to maintain the equilibrium between the concentration of surfactant molecules present at the droplets interface and in the continuous phase, some amount of Lubrizol U desorbs from the droplets, resulting in their coalescence and subsequent bigger final capsule size. This effect is not significant at low concentrations of used TDI. However, the final particles were almost 2 times larger than the size of the initial droplets when the HMDA to TDI molar ratio was 1:2. From the TEM images it could be also seen that the polyurea particles have a low contrast interior surrounded by a high contrast (dark) polymeric shell (see Figure 4a,b). The capsule shell thickness usually varies in the range between 15 and 30 nm and shows the tendency to increase with increasing amount of TDI introduced into the system. In the case of using high amounts (53) Borsus, J. M.; Jerome, R.; Teyssie, P. J. Appl. Polym. Sci. 1981, 26(9), 3027– 3043.
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Figure 4. TEM images (from cyclohexane phase) of polyurea capsules or frazzles prepared at different HMDA/TDI molar ratios: (a) 1:2, (b) 1:1.5, (c) 2:1.
of HMDA compared to the TDI (e.g., 1:0.4; 1.5:1, or 2:1 molar ratio), the DLS measurement data reveal different and not reproducible size fractions with broad polydispersity values (PDI > 0.5). This can be explained by the main production of low-molecular-weight polymeric chains due to unreacted amino groups, which does not lead to the formation of compact capsule structure. The absence of the intact capsules and strong polymer aggregation was also confirmed by the TEM studies (see Figure 4c). From Table 1, the DLS values are presenting the hydrodynamic diameter, whereas the size of dry capsules was determined using TEM. The DLS values measured in cyclohexane phase are slightly higher compared to those measured in an aqueous phase after redispersion step. The reason for this observation could be assigned to the high mobility of the hydrophobic chains originated from the Lubrizol U molecules that are attached onto the surface. This assumption was confirmed by TEM studies of the polyurea capsules prepared from the cyclohexane phase. The mean diameter of the capsules is slightly smaller due to the drying effect but comparable with the DLS values measured in the aqueous phase, when the hydrophobic chains are strongly adsorbed/collapsed onto the capsule surface. Thermogravimetric analysis was performed on polymeric capsules obtained at a HMDA/TDI molar ratio of 1:1, 1:1.5, and 1:2 in order to examine the thermal properties of the polymeric wall. The weight loss at ∼287 °C was observed for all samples, indicating high thermal stability of the produced polyurea capsules. 3.2. Variation of the Surfactant (Lubrizol U) Amount. The surfactant is responsible for the stability of the miniemulsion droplets as well as the resulting nanocapsules. It decreases the interfacial tension of the droplets and prevents their coagulation. The size of the capsules can be adjusted by performing the reaction in the presence of different surfactant amounts. The influence of Lubrizol U concentration on the final capsule size and shell thickness was studied by varying the surfactant weight ratio in cyclohexane. The amount of monomers was kept constant at a HMDA/TDI molar ratio of 1:1.5; this means that a considerable amount of isocyanate groups can be consumed for the reaction with amine groups of the surfactant. The average size of polyurea nanocapsules was determined by DLS, and the obtained results are plotted in Figure 5. Selected TEM images of polyurea nanocapsules manufactured with different amounts of Lubrizol U are presented in Figure 6. The obtained results show that with increasing surfactant concentration from 0.075 to 1.0 wt % the capsule size rapidly decreases from 1240 to 190 nm. This is in agreement with the theoretical approximations. About 15 mg of Lubrizol U (0.15 wt % on continuous phase) is required for complete coverage of the droplets, although stable miniemulsion (for at least 1.5 h) was produced with 25 mg (0.25 wt % on continuous phase). Furthermore, to get the 12088 DOI: 10.1021/la9017097
Figure 5. Influence of the Lubrizol U concentration on the capsule size; the HMDA/TDI molar ratio is 1:1.5. DLS values of polyurea capsules dispersed in cyclohexane.
information about the coverage of miniemulsion droplets with Lubrizol U, the interfacial tension between the miniemulsion and water was determined via spinning drop. For pure cyclohexane/ water system the interfacial tension was determined to be 48 mN/ m (in the literature 50 mN/m54). In the case of saturated Lubrizol U/cyclohexane solution the tension decreased up to 5.6 mN/m, which was taken as a saturation value for system containing inverse micelles. The interfacial tension of miniemulsion/water was measured to be 35 mN/m, therefore indicating the absence of micelles and incomplete coverage of the droplets with Lubrizol U molecules. In the concentration range between 1.0 and 2.0 wt % the size changes negligibly, and only PDI value increases. However, as the amount of Lubrizol U increases further, a strong aggregation of the capsules was observed and dispersion shows instability. We assume that the main reason for this might be due to the fact that the critical micelle concentration (cmc) of Lubrizol U is reached. Although in the presence of 1 wt % Lubrizol U the mean diameter of the capsules was smaller, the amount of Lubrizol U corresponded to 0.25 and 0.5 wt % was found to be optimal in order to prepare narrow sized (PDI e 0.15) polyurea nanocapsules. In this case, the capsule size was around 250 nm (after redispersion in water) and the shell thickness between 15 and 20 nm. 3.3. Polymer Formation Characterized by FT-IR Spectroscopy. To estimate the amount of TDI that was converted into a polymer, FT-IR measurements were carried out on freeze-dried samples of nanocapsules. The FT-IR spectra taken at different reaction times are shown in Figure 7a. (54) D’Ans, J.; Lax, E. Taschenbuch f€ ur Chemiker und Physiker, 4th ed.; SpringerVerlag: Berlin, 1992; Vol. I.
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Figure 6. TEM images of polyurea nanocapsules or frazzles obtained with different amounts of Lubrizol U (corresponding to the continuous phase): (a) 0.075, (b) 0.5, and (c) 2.5 wt %. HMDA/TDI molar ratio is 1:1.5.
Figure 7. FTIR spectra of freeze-dried polyurea capsules prepared from cyclohexane phase: (a) conversion kinetics of toluene 2,4diisocyanate during 24 h (HMDA/TDI molar ratio is 1:1.5); (b) after transferring into aqueous phase.
Figure 8. Hydrolysis of isocyanate group in the aqueous phase to form amine groups.
The spectra reveal a strong broad band ranging from about 3450 to 3200 cm-1 that usually corresponds to the combination of the stretching vibration bands of N-H groups in amine or imine bonds of the polyurea as well as the surfactant Lubrizol U. The adsorption band between 2950 and 2750 cm-1 corresponds to the C-H stretching vibrations in CH, CH2, and CH3 groups. From the features of the NdCdO peak, which appears at 2275 cm-1, it can be seen that the conversion of TDI is almost completed after 2 h of the reaction. The presence of a small residual peak of the isocyanate group which is visible after the reaction can be attributed to the groups, which are incorporated into the capsule shell only by one-side reacting with HMDA. After transferring the capsules into an aqueous phase, the isocyanate peak totally disappears (see Figure 7b), as a fact of the hydrolysis reaction occurring between isocyanate groups and water to form amine groups via formation of an unstable carbamic acid intermediate53 (Figure 8). This reaction occurs also during the capsule formation however to a low extent due to the presence of amine groups which are more reactive compared to the water hydroxyls. 3.4. Determination of the Lubrizol U Amount Bounded Covalently in the Polymeric Shell. The presence of amine groups in the molecule of Lubrizol U allows incorporation of the surfactant into the capsule’s shell under the polyaddition reaction. For example, one side of TDI molecule can react with the amine group of Lubrizol U, whereas the other isocyanate group of the Langmuir 2009, 25(20), 12084–12091
molecule can react with the primary amine group of HMDA. In the final capsule, Lubrizol U is then covalently bounded in the outer polymeric layer, stabilizing the capsule with its hydrophobic isobutylene chain oriented into the organic phase. Although the reaction of primary amine groups is known to be faster in comparison to the secondary ones (∼100 times), it could be possible that several amine groups from the Lubrizol U molecule participate in the polyaddition reaction with TDI. As a result, the formation of nanocapsules might be performed without the presence of HMDA. To prove this hypothesis, formation of nanocapsules at the droplet’s interface was carried out with different amounts of Lubrizol U and without addition of HMDA. It was found that the capsules are formed when the concentration of surfactant exceeds 200 mg (2 wt % on continuous phase). The amount of Lubrizol U that was reacted with TDI during the polymerization and the residual monomer concentration of TDI after the polyaddition reaction were estimated from the results of GPC analysis. The following molar ratios were studied: Lubrizol U/TDI 1:3.4; 1:1.7; 1:1; 1:0.8; 1:0.4 (or Lubrizol U þ HMDA/TDI 1:1.42; 1:1.35; 1:1.27; 1:1.21; 1:1.01), keeping the other parameters constant. The obtained results demonstrate that independently from the initial concentration of Lubrizol U, the total amount of the surfactant was covalently bounded into the polyurea shell. Additionally, the FTIR spectra of the polyurea capsules synthesized with different Lubrizol U amounts (25 and DOI: 10.1021/la9017097
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Rosenbauer et al. Table 2. Composition and Encapsulation Efficiency of Polyurea Nanocapsulesa
HMDA/TDI molar ratiob
fluorescein solution,c mg
PBS, mg
fluorescein,d mol
encapsulation efficiency,d %
95 375 375 2.8 10-8 95 250 500 1.9 10-8 -8 97 375 375 2.8 10 98 500 250 3.8 10-8 96 750 0 5.6 10-8 -8 97 1:2 (1:1.94) 375 375 2.8 10 a Each sample contains 10 g of cyclohexane with 0.25 wt % of dissolved Lubrizol U as a continuous phase. The amount of dispersed phase was kept constant at 750 mg. b The values in parentheses correspond to the HMDA þ Lubrizol U/TDI molar ratio. c Corresponds to a stock solution with a concentration of 7.5 10-5 mol L-1. d Corresponds to the samples of polyurea capsule after redispersion in the SDS aqueous solution (0.02 mol L-1, pH 7.0).
1:1 (1:0.97) 1:1.5 (1:1.46)
400 mg) reveal that the NdCdO peak (at 2275 cm-1) is lower in the case of using higher amounts of Lubrizol U (data not shown). However, due to the presence of -NH and -NH2 groups in Lubrizol molecule as well as in the reaction product, it is not possible to differentiate precisely how many and which groups participated in the reaction. Therefore, we compared the shell permeability of the capsules synthesized in the presence of Lubrizol U and with block copolymer surfactant, i.e., (butylene-co-ethylene)-b-(ethylene oxide) (see Encapsulation Efficiency section). The results of GPC analysis also provide the evidence that TDI is almost completely (more than 97 wt %) consumed during the reaction. Therefore, final capsules can be immediately utilized after the synthesis without performing any deactivation or purification step. With the molar excess of TDI (HMDA/TDI = 1:1.5 or 1:2) the functional diisocyanate groups react covalently either with one, alternatively with two HMDA functional groups as well as with a amine group from the Lubrizol U. Both ratios result in the formation of stable nanocapsules, and the only one difference is the average size (see Table 1). Larger capsules 440 nm vs 280 nm (measured in cyclohexane) are produced at HMDA/TDI = 1:2. 3.5. Encapsulation Efficiency. The encapsulation efficiency was studied by using the fluorescent dye as a hydrophilic marker. Fluorescein was dissolved in the disperse phase, and the interfacial polyaddition reaction was carried out resulting in fluorescent polyurea capsules. The determination of nonencapsulated fluorescein amount was performed on nanocapsule samples dispersed in cyclohexane (directly after polymerization) and in aqueous phase (after redispersion in SDS solution). Although from the hydrophilic nature of the fluorescein it is clear that its molecules will preferably stay inside the miniemulsion droplets before polymerization, the possibility of solubilization by the surfactant aggregates should not be excluded as well. Because of the low solubility of fluorescein in the cyclohexane, it was not possible to measure the amount of nonencapsulated fluorescein directly in the supernatant obtained after centrifugation of the capsules. Therefore, the capsule dispersion was freezedried first in order to remove the cyclohexane, substituted with a known amount of SDS aqueous solution (0.02 mol L-1 at pH 7.0), and then stirred for several hours in order to recover all free fluorescein molecules. Afterward, the capsules were centrifuged down, and the supernatant was examined with a fluorescence spectrometer on the presence of fluorescein. The fluorescence of supernatant obtained after centrifugation of polyurea capsules redispersed in aqueous phase was measured as well. One could expect that during the redispersion step the usage of sonication might result in the breakage some of the capsules and therefore affect the final fluorescein concentration in the continuous phase. By measuring the intensity of the fluorescein dye in the supernatant, the effectively encapsulated molar amount of dye can be 12090 DOI: 10.1021/la9017097
calculated, knowing the exact concentration of dye at certain intensity from the calibration curve. The calibration curve was evaluated from the defined concentrations of the fluorescein dye in the SDS aqueous solution (0.02 mol L-1 at pH 7.0). The influence of different monomer ratio on the encapsulation efficiency was studied at 0.25 wt % of Lubrizol U. The fluorescence measurements performed on freeze-dried samples after substitution with SDS aqueous solution showed very low intensities, confirming that the amount of nonencapsulated fluorescein in the cyclohexane phase is negligible (less than 1 wt %). However, it is worth to mention that due to the turbidity of the supernatant, the experimental error of the measurements was relatively high. This was not the case for supernatants obtained after centrifugation of redispersed polyurea capsules. Furthermore, from the control experiment it was found that after transferring the nanocapsules into the aqueous phase, the surface adsorbed fluorescein molecules (if any) desorbs into the continuous (aqueous) phase. The results of fluorescence measurements are summarized in Table 2. As it can be seen from the Table 2, an encapsulation efficiency of more than 95% was achieved, indicating that the obtained nanocapsules are composed of a stable and impermeable polymeric shell. For comparison, the encapsulation efficiency experiments were performed with polyurea capsules stabilized with block copolymer surfactant (butylene-co-ethylene)-b-(ethylene oxide) described previously.45 It was found that after several washing steps more than 50 wt % of the encapsulated material leaked out. In contrast, the leakage was less than 10% in the case of using Lubrizol U, confirming that degree of the shell cross-linkage is higher in contrast to (butylene-co-ethylene)-b-(ethylene oxide). 3.6. Uptake of Polyurea Capsules into the HeLa Cells. In order to evidence one of the potential applications of the nanocapsules, for example, in a biomedical field, the fluorescein-containing polyurea capsules were used as markers in the cellular uptake experiments. The nanocapsules redispersed in the SDS aqueous solution were added to the HeLa cells, and after 24 h incubation the uptake efficiency was detected using fluorescent confocal microscopy (see Figure 9). The experiments confirmed that the capsules are not causing the cell death (more than 96% of the cells stay vital) and were efficiently internalized into the cell and not simply attached to the cell membrane. 3.7. Encapsulation of Magnetite. As was shown above and in the previous publications of our group, the miniemulsion technique allows efficient entrapment of small organic molecules (e.g., fluorescein, as shown here), water-soluble complexes,46 and shear-sensitive DNA molecules40 into the aqueous core nanocapsules. The presence of magnetite nanoparticles inside a capsule offers promising drug delivery applications. In this regard, magnetite nanoparticles of 10 nm dispersed in aqueous phase and stabilized with SDS were used as a core material in order to synthesize the magnetic polyurea nanocapsules. The reaction was performed as described above, via interfacial polyaddition at the molar ratio of HMDA/TDI 1:1.5 and 50 mg of Lubrizol U. Langmuir 2009, 25(20), 12084–12091
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The images reveal irregularly shaped capsules and the presence of magnetite nanoparticles within a polymeric matrix. From the scanning electron microscopy images (not shown) no magnetite was found to be adsorbed onto the capsules surface. It could also be seen that not all capsules are filled with magnetite and that the distribution of magnetite within a capsule is not homogeneous. These could be improved by variation of the sonication parameters or using higher initial concentrations of surface modified (in order to avoid aggregation of magnetic nanoparticles) magnetite dispersion. Related experiments are now in progress, and the results will be presented in a separate paper.
Figure 9. Confocal fluorescent microscopy of HeLa cells after the uptake of polyurea capsules (green). The capsules were added to the HeLa cells in a concentration of 75 μg mL-1.
Figure 10. TEM images of magnetic polyurea nanocapsules (from cyclohexane phase).
The hydrodynamic diameter and the PDI value of the obtained capsules were within the size range of the polyurea capsules prepared without magnetite (Dz = 273 nm, PDI = 0.14), indicating that there is no influence of the magnetite on the interfacial polymerization process. The amount of encapsulated magnetite was estimated from the TGA curve performed with the freeze-dried capsules. The obtained results demonstrate that about 85% of the introduced magnetite was encapsulated, that corresponds to 0.11 gmagnetite per gpolymer. The morphology of nanocapsules was studied by TEM (see Figure 10).
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4. Conclusion Polyurea nanocapsules consisting of an aqueous core were synthesized by a polyaddition reaction at the inverse miniemulsion droplet interface. The use of amino-functionalized surfactant (Lubrizol U) can improve the stability and impermeability of the final capsules through the formation of the covalent bonds between surfactant and polymeric shell. The FT-IR data obtained from the kinetic experiments indicate that the conversion of TDI is almost completed after 2 h of the reaction. The obtained capsules were characterized for their average size, shell thickness, morphology, and loading efficiency when produced with different monomer molar ratio and surfactant concentration. It was found that stable and well-defined polymeric capsules within a size range between 255 and 440 nm were obtained when the amount of HMDA (including Lubrizol U) was equal or less in molar ratio compared to the TDI concentration. The capsule shell thickness varies in the range between 15 and 30 nm, showing a tendency to increase with increasing amount of TDI introduced into the system. The encapsulation efficiency about 95% and 85% was achieved with water-soluble fluorescent dye molecules and magnetite aqueous dispersion, respectively, thus indicating the effectiveness and flexibility of the proposed miniemulsion approach. Looking for a potential application, as an example, the fluorescein-containing polyurea capsules redispersed in the SDS aqueous solution were used as markers in the cell uptake studies. The obtained results confirmed that the capsules are not toxic and were efficiently internalized into the cell. Acknowledgment. This research was supported by German Research Foundation (DFG). The authors thank Julia Dausend and Dr. Volker Mail€ander for help in cell uptake experiments and discussions.
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