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Langmuir 2007, 23, 9546-9550
High-Density Encapsulation of Fe3O4 Nanoparticles in Lipid Vesicles Andy Wijaya§ and Kimberly Hamad-Schifferli*,†,‡ Department of Mechanical Engineering, Department of Biological Engineering, and Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 ReceiVed April 17, 2007. In Final Form: July 17, 2007 We report a morphological study of the encapsulation of 12-nm Fe3O4 nanoparticles (NPs) in large unilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC). Preparation was done by reverse-phase evaporation. Phase behavior of the NP-lipid system was studied so that the loading of NPs in vesicles could be maximized. Increasing NP concentration significantly affects the resulting lipid morphology in a manner similar to increasing lipid concentration. Optimal production of high-density NP-loaded vesicles (HNLVs) requires temperatures of 50 °C, higher than the main phase transition (Tm) of DPPC. The formation of fully enclosed HNLVs requires incubation times of at least hours.
Introduction Liposomes have attracted interest for drug delivery applications because of their ability to encapsulate and release payloads.1 Because their composition is similar to that of cell membranes and their surface chemistry can be modified for specific targeting, they have unique biological capabilities.2 For example, they have been used to target tumor sites and release anticancer drugs.3 Encapsulation of nanoparticles (NPs) in liposomes is desirable for applications such as triggered release due to hyperthermia, drug delivery,4-9 enhancing magnetic resonance imaging,10 imaging by fluorescent NPs,11 and photothermal therapy.12 Liposomes are self-assembled vesicles of amphiphilic lipid molecules. Currently, numerous preparation methods of lipid vesicles exist.13,14 Encapsulation of hydrophilic moieties is achieved by introduction to the aqueous phase so that they can be spontaneously captured inside the liposomes during vesicle formation. Approaches that increase the internal liposome volume have been pursued for the encapsulation of large amounts of payloads. The reverse-phase evaporation (REV) method by Szoka et al.15 has been successful in creating large unilamellar vesicles (LUVs) with a large internal aqueous space, and has been used to make liposomes that encapsulate molecules, proteins, DNA,16 * Corresponding author. E-mail:
[email protected]. † Department of Mechanical Engineering. ‡ Department of Biological Engineering. § Department of Chemical Engineering. (1) Weinstein, J. N.; Klausner, R. D.; Innerarity, T.; Ralston, E.; Blumenthal, R. Biochim. Biophys. Acta 1981, 647 (2), 270-284. (2) Torchilin, V. P. Nat. ReV. Drug DiscoVery 2005, 4 (2), 145-160. (3) Weinstein, J. N.; Magin, R. L.; Yatvin, M. B.; Zaharko, D. S. Science 1979, 204 (4389), 188-191. (4) Yatvin, M. B.; Weinstein, J. N.; Dennis, W. H.; Blumenthal, R. Science 1978, 202 (4374), 1290-1293. (5) Viroonchatapan, E.; Sato, H.; Ueno, M.; Adachi, I.; Tazawa, K.; Horikoshi, I. Life Sci. 1996, 58 (24), 2251-2261. (6) Gaber, M. H.; Wu, N. Z.; Hong, K. L.; Huang, S. K.; Dewhirst, M. W.; Papahadjopoulos, D. Int. J. Radiat. Oncol. 1996, 36 (5), 1177-1187. (7) Kong, G.; Dewhirst, M. W. Int. J. Hyperthermia 1999, 15 (5), 345-370. (8) Needham, D.; Anyarambhatla, G.; Kong, G.; Dewhirst, M. W. Cancer Res. 2000, 60, 1197-1201. (9) Needham, D.; Dewhirst, M. W. AdV. Drug DeliVery ReV. 2001, 53, 285305. (10) Bulte, J. W. M.; Cuyper, M. D. Methods Enzymol. 2003, 373, 175-198. (11) Chen, C.-S.; Yao, J.; Durst, R. A. J. Nanopart. Res. 2006, 8, 1033-1038. (12) Kasili, P. M.; Vo-Dinh, T. Nanobiotechnology 2005, 1 (3), 245-252. (13) Szoka, F.; Papahadjopoulos, D. Annu. ReV. Biophys. Bioeng. 1980, 9, 467-508. (14) Woodle, M. C.; Papahadjopoulos, D. Methods Enzymol. 1989, 171, 193217. (15) Szoka, F., Jr.; Papahadjopoulos, D. Proc. Natl. Acad. Sci. U.S.A. 1978, 75 (9), 4194-4198. (16) Fenske, D. B.; Cullis, P. R. Methods Enzymol. 2005, 391, 7-40.
and NPs.11,17-19 The mechanism of encapsulation is hypothesized to occur when lipid vesicles self-assemble from a collapsed gellike state, enclosing the payload into a vesicle (Figure 2, panels 5 and 6).15,20 For many applications of NP-encapsulated liposomes, highdensity encapsulation is desirable. Hyperthermia and drug delivery both benefit from high NP loading. Also, magnetic resonance contrast enhancement relies on NP clustering and requires that the NPs be fairly close together, where interparticle distances are on the order of only a few NP diameters. Generally, it is difficult to achieve uniformly spherical liposomes with high loading of NPs,17-19,21-25 and often low yields result. Recently, Chen et al.11 reported, on average, only three quantum dots per liposome of d ∼ 50-300 nm. Martina et al.26 reported ∼60 Fe2O3 NPs/100 nm liposome. While liposome formation by itself is well understood, phase diagrams have not been constructed in the case where an encapsulant with significant volume is present. It is expected that the presence of NPs, especially at high concentrations, will appreciably affect liposome phase diagrams, such as for block copolymers.27 Therefore, understanding NPlipid morphologies is necessary to optimize NP encapsulation. We performed a systematic study of the phase behavior of dipalmitoylphosphatidylcholine (DPPC) with 12 nm Fe3O4 NPs, with the goal of maximizing the NP encapsulation in DPPC vesicles. Transmission electron microscopy (TEM) imaging was used to qualitatively analyze resulting morphologies and quantify NP-loaded liposome size distribution. We are able to synthesize liposomes that are densely packed with NPs. Varying lipid and NP concentration results in different NP-lipid morphological (17) Viroonchatapan, E.; Ueno, M.; Sato, H.; Adachi, I.; Nagai, H.; Tazawa, K.; Horikoshi, I. Pharm. Res. 1995, 12 (8), 1176-1183. (18) Hong, K.; Friend, D. S.; Glabe, C. G.; Papahadjopoulos, D. Biochim. Biophys. Acta 1983, 732 (1), 320-323. (19) Babincova, M.; Sourivong, P.; Chorvat, D.; Babinec, P. J. Magn. Magn. Mater. 1999, 194 (1-3), 163-166. (20) Lasic, D. D. Biochem. J. 1988, 256, 1-11. (21) Decuyper, M.; Joniau, M. Eur. Biophys. J. 1988, 15 (5), 311-319. (22) Sangregorio, C.; Wiemann, J. K.; O’Connor, C. J.; Rosenzweig, Z. J. Appl. Phys. 1999, 85 (8), 5699-5701. (23) Kuckelhaus, S.; Reis, S. C.; Carneiro, M. F.; Tedesco, A. C.; Oliveira, D. M.; Lima, E. C. D.; Morais, P. C.; Azevedo, R. B.; Lacava, Z. G. M. J. Magn. Magn. Mater. 2004, 272-276, 2402-2403. (24) Giri, J.; Thakurta, S. G.; Bellare, J.; Nigam, A. K.; Bahadur, D. J. Magn. Magn. Mater. 2005, 293 (1), 62-68. (25) Gonzales, M.; Krishnan, K. M. J. Magn. Magn. Mater. 2005, 293, 265270. (26) Martina, M.-S.; Fortin, J.-P.; Me´nager, C.; Cle´ment, O.; Barratt, G.; Grabielle-Madelmont, C.; Gazeau, F.; Cabuil, V.; Lesieur, S. J. Am. Chem. Soc. 2005, 127, 10676-10685. (27) Park, M. J.; Park, J.; Hyeon, T.; Char, K. J. Polym. Sci., Part B: Polym. Phys. 2006, 44, 3571-3579.
10.1021/la701128b CCC: $37.00 © 2007 American Chemical Society Published on Web 08/16/2007
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Langmuir, Vol. 23, No. 19, 2007 9547 temperature was well above the Tm of DPPC (41.5 °C). Other REV methods to prepare DPPC vesicles15 and magnetoliposomes17 have used T ) 20-25 °C and 42 °C, respectively, while others use T ) 45-50 °C.11,19,29 Here, sample repeatability was greatly improved by operating at T ) 50 °C, which is significantly higher than the Tm of DPPC. Therefore, all data presented on samples here had a preparation temperature of 50 °C. The vacuum in the flask was broken, and purified deionized water was added to the gel (or continuous-phase water, CPW30) to replenish any that was lost during evaporation, or for further dilution to achieve certain final concentrations. The amount of CPW varied from synthesis to synthesis, and may also depend on the evaporator setup. Pressure was further reduced at increments of -1 inHg/5 min until reaching P ) -22 inHg. At this point, the gel had completely collapsed, forming lipid vesicles (panel 6). This pressure was maintained for another 15 min to remove traces of solvent. The resulting aqueous solution was allowed to sit undisturbed for at least 3 h before deposition on a TEM grid for imaging. Purification by Centrifugation. To remove aggregates, the solution was first centrifuged at 100g for 5 min, and the supernatant was retained. The solution was then centrifuged at 200g for 5 min to remove unencapsulated materials. The supernatant containing nonencapsulated NPs and calcein was discarded, and the desired product in the precipitate was resuspended in 20 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) buffer solution (pH 7). Centrifugation was repeated twice, resulting in a purified solution of NP-encapsulated liposome vesicles. Imaging and Sizing. Samples were imaged by TEM (JEOL 2011). Samples were deposited on holey carbon grids (Ted Pella) directly from solution. Images were taken at least within 3 days. Size analysis was done by Image J.31
Figure 1. (a) TEM image and (b) size distribution of Fe3O4 NPs. 〈d〉 ) 12.5 ( 3 nm, 651 NPs sized.
structures. We find that high NP concentrations perturb the phase diagram in such a way that it effectively increases the concentration of the lipid. Key parameters for producing high-yield NPloaded liposomes are determined. Experimental Section Aqueous Fe3O4 NPs were purchased from FerroTec (EMG 705). The NPs were at a volume fraction of 3.9% and used without modification except dilution. TEM imaging (Figure 1a) confirmed that NPs were well dispersed and not aggregated, with 〈d〉 ) 12.5 ( 3.4 nm (Figure 1b). DPPC was purchased from Avanti Lipids. All other chemicals were purchased from Sigma-Aldrich. Liposome Preparation. To produce liposomes with large internal volume and thus potential for high loading, we used a slightly modified version of the reverse-evaporation method15 (protocol in Figure 2). Here, 20, 40, or 60 mg of DPPC was dissolved in 12 mL of a v/v ) 1:1 mixture of chloroform and isopropyl ether in a 50 mL round-bottom flask that was cleaned using the Avanti procedure.28 This is referred to as the “organic solution” (panel 1). Then, 1.5 mL of 3.9, 1.95, 0.98, or 0.39 vol % Fe3O4 NP solution and 1.5 mL of 20 mM calcein (as a model molecule for encapsulated drug) at pH 7 were added into the organic solution (referred to as the “mixture”, panel 2). Ar (g) was bubbled through the mixture to remove any oxygen. The mixture was sonicated in a bath sonicator at T ) 50 °C for 5 min to produce a water-in-oil emulsion (Figure 2, panel 3). The mixture was cooled slowly and sat for >30 min. The stability of the emulsion was confirmed before proceeding. The organic solution was removed from the emulsion slowly using a rotary evaporator at T ) 50 °C with P ) -11 to -14 inHg for 2.5 h (panel 4). At this point, most of the solvent had been removed, forming a gel, which adhered to the flask wall (panel 5). Note that the operating (28) AvantiLipids Homepage. GlasswareCleaningProcedure.asp.
http://www.avantilipids.com/Technical
Results and Discussion Concentration Effect. By varying the lipid and NP starting concentrations and the amount of added CPW, we studied the effect of lipid and NP concentration on the resulting lipid/NP morphology. There is a limit to increasing the lipid concentration, because too high of a lipid concentration will result in the inability of the gel (Figure 2, panel 5) to collapse and form vesicles; this remains stuck to the flask wall and is not recoverable. In contrast, starting lipid concentration must be high enough to produce a stable water-in-oil emulsion (Figure 2, panel 3). Figure 3a shows the range of lipid and NP concentrations explored. Figure 3b-f shows images of the corresponding morphological features. The lipids are unstained, so dark areas in the image correspond to NPs. These images were taken of the original solutions at least 3 h after preparation, without purification by centrifugation. These morphologies are identical to those observed for the lipid-only system.32 At the highest NP concentration (∼100 mg/mL, squares in Figure 3a), perforated bicelles are formed (Figure 3b). The NPs are dispersed in the bicelle sheet that has pores (resembling a slice of Swiss cheese). Some of these bicelle sheets contain features of spherical lipid vesicles (resembling bubble wrap, Supporting Information). A mixture of both was commonly observed. Others have proposed the structural model of these perforated bicelles (or perforated lamellar phase).33-37 (29) Duzgunes, N.; Wilschut, J.; Hong, K.; Fraley, R.; Perry, C.; Friend, D. S.; James, T. L.; Papahadjopoulos, D. Biochim. Biophys. Acta 1983, 732 (1), 289-299. (30) Pidgeon, C.; McNeely, S.; Schmidt, T.; Johnson, J. E. Biochemistry 1987, 26 (1), 17-29. (31) Abramoff, M. D.; Magelhaes, P. J.; Ram, S. J. Biophotonics Int. 2004, 11 (7), 36-42. (32) Nieh, M. P.; Raghunathan, V. A.; Kline, S. R.; Harroun, T. A.; Huang, C. Y.; Pencer, J.; Katsaras, J. Langmuir 2005, 21 (15), 6656-6661. (33) Nieh, M. P.; Glinka, C. J.; Krueger, S.; Prosser, R. S.; Katsaras, J. Langmuir 2001, 17 (9), 2629-2638. (34) Gaemers, S.; Bax, A. J. Am. Chem. Soc. 2001, 123 (49), 12343-12352.
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Figure 2. Method for NP encapsulation into liposome vesicles via the REV method: lines: lipid; dark gray: organic solvent; light gray: water; circles: NPs; stars: calcein. (1) Lipid is dispersed in organic solvent. (2) NPs and calcein are introduced in water. (3) Mixture is sonicated to make an emulsion. (4) Organic solvent is evaporated off. (5) Evaporation continues until a gel-like state is formed. (6) Further evaporation and addition of water causes spontaneous formation of lipid bilayer vesicles containing NPs and calcein.
Figure 3. (a) Plot showing lipid and NP concentrations used. (b-f) TEM images of the corresponding morphologies: (b) holey bilayer sheet; (c) mixed bilayer sheet with NP-loaded vesicles; (d) mixed empty and NP-loaded vesicles; (e) mostly HNLV; (f) mostly lower density-loaded or smaller vesicles.
Phase behavior studies of lipid-only systems find that spherical LUVs and multilamellar vesicles are formed at low lipid concentrations, while the perforated lamellar phase occurs at high concentrations.32,33,38 Clearly, the NP concentration is high enough to affect the lipid phase diagram significantly, and changes the resulting phases. For example, increasing NP concentration at a fixed lipid concentration, the resulting morphology moves from high-density NP-loaded vesicles (HNLVs) to perforated (35) Tan, C. B.; Fung, B. M.; Cho, G. J. J. Am. Chem. Soc. 2002, 124 (39), 11827-11832. (36) Loudet, U.; Khemtemourian, L.; Aussenac, F.; Gineste, S.; Achard, M. F.; Dufourc, E. J. Biochim. Biophys. Acta 2005, 1724 (3), 315-323. (37) Almgren, M. J. Dispersion Sci. Technol. 2007, 28 (1), 43-54. (38) Nieh, M. P.; Glinka, C. J.; Krueger, S.; Prosser, R. S.; Katsaras, J. Biophys. J. 2002, 82 (5), 2487-2498.
lamellae. This effect is similar to increasing lipid concentration in lipid-only systems. For example, we can obtain the perforated bicelle phase at low lipid concentrations simply by increasing the NP concentration. This suggests that, in NP-lipid systems, concentrations of both the NPs and lipids will affect the resulting morphologies. Interestingly, similar effects have been observed for lipids in the presence of large molecular weight hyaluronan, indicating that the introduction of large non-lipid species can favor the formation of lamellae.39 When the NP concentration is above some threshold, varying the lipid concentration from 15 to 40 mg/mL has little effect on the resulting phase (squares and diamonds, Figure 3a), resulting only in morphologies (39) Pasquali-Ronchetti, I.; Quaglino, D.; Mori, G.; Bacchelli, B.; Ghosh, P. J. Struct. Biol. 1997, 120 (1), 1-10.
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Figure 4. TEM images of (a) HNLVs and (b) low-density NPloaded or smaller vesicles.
favored by high lipid concentrations. This indicates that, in this regime of the NP-lipid phase diagram, the effective lipid behavior is dominated by the NP concentration. This is most likely due to the fact that, above this concentration, the NP volume fraction becomes comparable to that of the lipid. For a fixed lipid concentration of 23-27 mg/mL, decreasing the NP concentration below 101-105 mg/mL results in different morphologies. At [NP] ) 54-58 mg/mL (diamonds, Figure 3a), a mixture of perforated bicelles and NP-loaded vesicles resulted (Figure 3c). Decreasing [NP] to 20 mg/mL (triangles, Figure 3a) yields a mixture of empty vesicles and NP-loaded vesicles (Figure 3d). In the image, unloaded vesicles appear as large empty circular structures. Finally, at [NP] )12-13 mg/mL (circles, Figure 3a), mostly spherical vesicles with high loading of NPs are formed (Figure 3e), or HNLVs. Figure 4a shows a high-resolution image of an HNLV ∼200 nm in diameter, which contains a spherical mass of NPs at high density. At lower NP concentrations (5-13 mg/mL), changing the lipid concentration varies vesicle loading and size (circles and stars, Figure 3a). Lower lipid concentrations yields vesicles that could be either vesicles loaded at a lower density or smaller vesicles loaded at the same density as the HNLV (Figure 3f). Figure 4b shows a higher resolution image of one of these liposomes that is ∼100 nm in diameter. This could be due to the fact that, at lower lipid concentrations, only smaller vesicles can be formed on average. At [lipid] ) 21-23 mg/mL, the obtained morphology is predominantly HNLV (Figure 3e), while, at [lipid] ) 7-9 mg/mL, it is predominantly lower density-loaded or smaller liposomes (Figure 3f). Since it is desirable to produce high-yield NP encapsulation into liposomes, the predominantly HNLV sample (Figure 3e) was purified by centrifugation to remove unencapsulated NPs
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Figure 5. (a) TEM image and (b) size distribution of centrifugepurified HNLVs; 〈d〉 ) 225 ( 67 nm, 388 HNLVs sized.
(Experimental Section). The resulting solution contained only uniformly spherical HNLVs with no free NPs (Figure 5a). Size distribution measured by TEM was d ) 225 ( 67 nm (Figure 5b). Kinetic Effects. Incomplete vesicle formation has been observed in molecular dynamics simulations, where incomplete closure of the vesicle results in a water pore in the membrane. Because the bilayer vesicle is not sealed, the hydrophilic contents can spill out.40 We observed instances of this phenomenon where vesicles that did not reach complete closure, and NPs can be seen leaking out (Figure 6). This image is of the same sample in Figure 3e and Figure 5, except the aliquot was withdrawn within 30 min after liposome preparation, as opposed to >3 h. This agrees with studies of the kinetic rate of micelle-to-vesicle transition of other lipid systems, which find that the time scale for the growth of disk-like intermediate micelles to closed vesicles is typically hours.41,42 Dynamic light scattering experiments on egg lecithin suspensions after sonication have shown that complete transformation from planar disks to closed vesicles was observed after 3 h.43 Other Effects. Centrifugation for long times (>10 min) or at higher speeds (g1000 g) induced vesicle fusion (Figure 7). Others have studied the fusion phenomenon of lipid vesicles during storage, which showed an apparent dependence on the duration of storage time and temperature.44-47 (40) Marrink, S. J.; Mark, A. E. J. Am. Chem. Soc. 2003, 125, 15233-15242. (41) Leng, J.; Egelhaaf, S. U.; Cates, M. E. Biophys. J. 2003, 85 (3), 16241646. (42) Egelhaaf, S. U.; Schurtenberger, P. Phys. ReV. Lett. 1999, 82 (13), 28042807. (43) Fromherz, P.; Ruppel, D. FEBS Lett. 1985, 179 (1), 155-159. (44) Larrabee, A. L. Biochemistry 1979, 18 (15), 3321-3326. (45) Schullery, S. E.; Schmidt, C. F.; Felgner, P.; Tillack, T. W.; Thompson, T. E. Biochemistry 1980, 19 (17), 3919-3923. (46) Mcconnell, D. S.; Schullery, S. E. Biochim. Biophys. Acta 1985, 818 (1), 13-22.
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Figure 6. (a) TEM image of a predominantly HNLV solution sampled within 30 min after liposome preparation showing that the majority of vesicles have incomplete closure. (b) High-resolution TEM images from panel a showing the incomplete vesicle closures where the NPs spill out.
Conclusion In summary, the resulting morphology of a lipid/NP system is strongly dependent on NP and lipid concentrations. This study has enabled the production of large spherical lipid vesicles that encapsulate 12 nm Fe3O4 NPs at high yield (HNLVs). The presence of NPs perturbs the phase diagram, with the effect of increasing the effective lipid concentration. Key factors for HNLV (47) Lentz, B. R.; Carpenter, T. J.; Alford, D. R. Biochemistry 1987, 26 (17), 5389-5397.
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Figure 7. (a) TEM image of an HNLV solution after 1000g centrifuge purification showing that the majority of HNLVs fused with each other. (b) High-resolution TEM images showing the fused lipid bilayer of two HNLVs.
formation include an operating temperature of 50 °C, which is higher than the lipid transition temperature. Sufficient time for complete vesicle formation is necessary (∼hours). Future studies will assess liposome stability, finer size control, and applicability for controlled release. Acknowledgment. We are grateful to the MIT Center for Materials Science and Engineering for TEM usage. Supporting Information Available: High-resolution images of some of the figures and additional high-resolution TEM images. This material is available free of charge via the Internet at http://pubs.acs.org. LA701128B
