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Magnetic Targeting and Cellular Uptake of Polymer Microcapsules Simultaneously Functionalized with Magnetic and Luminescent Nanocrystals Bernd Zebli,† Andrei S. Susha,† Gleb B. Sukhorukov,‡ Andrey L. Rogach,*,† and Wolfgang J. Parak*,† Physics Department & Center for NanoScience (CeNS), Ludwig-Maximilians-Universita¨ t Mu¨ nchen, D-80799 Munich, Germany, and Max Planck Institute of Colloids and Interfaces, D-14424 Potsdam, Germany Received January 26, 2005. In Final Form: March 11, 2005 By using a flow channel system for modeling the bloodstream in the circulatory system and by locally creating a magnetic field gradient caused by a permanent magnet, we demonstrate specific trapping of polymer capsules simultaneously functionalized with two types of nanoparticlessmagnetic and luminescent nanocrystals. In the regions where the capsules were trapped by the magnetic field, drastically increased uptake of capsules by cells has been observed. The uptake of capsules by cells could be conveniently monitored with a fluorescence microscope by the luminescence of CdTe nanocrystals that had been embedded into the shells of the capsules. Our experiments envisage the feasibility of magnetic targeting of polymer capsules loaded by pharmaceutical agents to pathogenic parts of a tissue.
Among a number of drug-delivery systems developed up to date,1 polymer microcapsules made by the layerby-layer absorption of oppositely charged macromolecules on colloidal templates2 represent a versatile system possessing multiple functionalities in a single entity. The use of functionalized polymer capsules as drug carrier systems, microreactors, and sensors is envisaged, as has been discussed in a recent review.3-5 Labeling of capsules by luminescent substances, for example, semiconductor nanocrystals6-8 (colloidally synthesized quantum dots9-11) provides a possibility to trace their pathways within a tissue, whereas loading them with magnetic nanoparticles8 allows for manipulation by an external magnetic field gradient. Magnetic drug targeting offers several attractive possibilities in biomedicine, allowing for the locoregional cancer treatment with reduced side effects.12 The magnetic drug carriers used nowadays usually comprise a magnetic * To whom correspondence should be addressed. E-mail:
[email protected] (A.L.R.);
[email protected] (W.J.P.). † Physics Department & Center for Nanoscience. ‡ Max Planck Institute of Colloids and Interfaces. (1) Haag, R. Angew. Chem., Int. Ed. 2004, 43, 278 and references therein. (2) Donath, E.; Sukhorukov, G. B.; Caruso, F.; Davis, S. A.; Mo¨hwald, H. Angew. Chem., Int. Ed. 1998, 37, 2202. (3) Peyratout, C. S.; Da¨hne, L. Angew. Chem., Int. Ed. 2004, 43, 3762. (4) Shchukin, D. G.; Sukhorukov, G. B. Adv. Mater. 2004, 16, 671. (5) Sukhorukov, G. B.; Rogach, A. L.; Zebli, B.; Liedl, T.; Skirtach, A. G.; Ko¨hler, K.; Antipov, A. A.; Gaponik, N.; Susha, A. S.; Winterhalter, M.; Parak, W. J. Small 2005, 1, 194. (6) Gaponik, N.; Radtchenko, I. L.; Sukhorukov, G. B.; Weller, H.; Rogach, A. L. Adv. Mater. 2002, 14, 879. (7) Gaponik, N.; Radtchenko, I. L.; Gerstenberger, M. R.; Fedutik, Y. A.; Sukhorukov, G. B.; Rogach, A. L. NanoLett 2003, 3, 369. (8) Gaponik, N.; Radtchenko, I. L.; Sukhorukov, G. B.; Rogach, A. L. Langmuir 2004, 20, 1449. (9) Parak, W. J.; Gerion, D.; Pellegrino, T.; Zanchet, D.; Micheel, C.; Williams, S. C.; Boudreau, R.; Le Gros, M. A.; Larabell, C. A.; Alivisatos, A. P. Nanotechnology 2003, 14, R15. (10) Pellegrino, T.; Kudera, S.; Liedl, T.; Mun˜oz Javier, A.; Manna, L.; Parak, W. J. Small 2005, 1, 48. (11) Parak, W. J.; Pellegrino, T.; Planck, C. Nanotechnology 2005, 16, R9. (12) Alexiou, C.; Arnold, W.; Klein, R.; Parak, F. G.; Hulin, P.; Bergemann, C.; Erhardt, W.; Wagenpfeil, S.; Lu¨bbe, A. S. Cancer Res. 2000, 60, 6641.
submicroparticle core coated with a biocompatible polymer, or a porous biocompatible polymer microparticle with inclusions of magnetic nanoparticles.13 These carriers can be coupled to cytotoxic drugs via several functional surface groups. In this work, we address the problem of magnetic targeting of polymer microcapsules simultaneously loaded with magnetic and luminescent nanoparticles, by demonstrating their accumulation and increased cellular uptake at specific places determined by a magnetic field gradient in a model flow channel system. The model system chosen in our experiments simulates an interplay of two forces: movement of the capsules under the flow and manipulation of capsules by a magnetic field gradient. The flow models the bloodstream in the circulatory system, while the magnetic field gradient allows for localization/ concentration of capsules loaded with magnetic nanoparticles at specific places. Magnetite (Fe3O4) nanoparticles with an average size of 10 nm, stabilized with tetramethylammonium hydroxide, were synthesized in water according to standard protocols.14 CdTe nanocrystals (3.5 nm diameter, emission maximum at 625 nm) used for luminescent labeling of the capsules were synthesized in aqueous solution according to a previously published procedure by employing thioglycolic acid as a stabilizer carrying negatively charged -COOH groups.15 The polymer microcapsules were templated on melamine formaldehyde cores (Microparticles GmbH, Berlin), 3 µm in diameter, which were solved afterward by treatment with 0.1 M HCl. The walls of the capsules were grown by applying a layer-by-layer coating3 and consisted of five double layers of oppositely charged polyelectrolytes: poly(styrene sulfonate) (PSS, Mw ∼ 70 000) and poly(allylamine hydrochloride) (PAH, Mw ∼ 50 000). The capsule walls were infiltrated with magnetite nanoparticles by soaking in a concentrated colloidal solution of the particles overnight, coated by additional layers of PAH, PSS, and PAH, and labeled by CdTe (13) Pankhurst, Q. A.; Connoly, J.; Jones, S. K.; Dobson, J. J. Phys. D: Appl. Phys. 2003, 36, R167. (14) Massart, R. IEEE Trans. Magn. 1981, 17, 1247. (15) Gaponik, N.; Talapin, D. V.; Rogach, A. L.; Hoppe, K.; Shevchenko, E. V.; Kornowski, A.; Eychmu¨ller, A.; Weller, H. J. Phys. Chem. B 2002, 106, 7177.
10.1021/la0502286 CCC: $30.25 © 2005 American Chemical Society Published on Web 04/16/2005
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Scheme 1. Model System for the Magnetic Delivery Experiments with Polymer Capsules Functionalized with Magnetic Nanocrystalsa
a The arrows indicate the flow of the cell medium with injected capsules modeling the bloodstream. The capsules are accumulated by trapping them in the magnetic field of a permanent magnet and are preferentially taken up by cells located in this region.
nanocrystals utilizing electrostatic attraction between the acidic groups of the nanoparticles’ stabilizer and the amino groups of PAH.6-8 Intermediate polymer layers between Fe3O4 and CdTe nanoparticles were necessary to reduce undesirable quenching effects of the luminescence of the latter. Scheme 1 shows the model flow channel system used in our experiments. Polymer capsules loaded with magnetic and luminescent nanoparticles were injected into a reservoir containing a Leibovitz L-15 cell medium with 2 mM L-glutamine supplemented with 0.01 mg/mL insulin (85%) and 15% fetal bovine serum, which was connected to a flow channel (ibidi Integrated Biodiagnostics, Munich, Germany) through a peristaltic pump. The velocity of the flow was regulated by the pump and varied between 5 and 16 cm/s in our experiments; the values were chosen to be comparable to the blood flow rates in the circulatory system. Both the flow channel and the cell medium reservoir were placed inside an incubator of a fluorescence microscope (Axiovert 200M, Zeiss) to maintain a constant temperature of 37 °C. Breast cancer cells (MDA-MB-435s) were cultured at the bottom of the flow channel, which was coated with a collagen to promote cell adhesion. About 5 × 104 cells were grown on a surface area of 250 mm2. Experiments were performed with a concentration of approximately 1.7 × 106 capsules/mL of cell medium. By taking into account the volume of the flow channel (100 µL), the number of capsules per cell in the flow channel was estimated to be between 3 and 4. Two cubic permanent magnets (NbFeB, 5 mm edge length, magnetic field strength of 1.3 T) were placed under the flow channel for 4 h to allow the capsules to accumulate in this particular region. After removal of the magnets and removal of capsules that have not been ingested by the cells, the number of capsules taken up by each cell was counted by applying fluorescence microscopy for 50 individual cells situated directly under the edge of the magnet. This number was compared with a similar statistics recorded for cells at positions 5.5 and 11 mm away from the edge of the magnet. Using a 100× oil-immersion objective, it was possible to distinguish between the capsules adherent on the cell surface and the internalized capsules by applying focus criteria as discussed in ref 5. Capsules loaded with magnetic nanoparticles in a resting cell medium align into parallel stripes in the magnetic field of a permanent magnet (Figure 1A). In a cell medium under flow, the capsules are captured by the magnetic field gradient and are accumulated in the flow channel at the area of the location of the magnet (Figure
Figure 1. (A) Alignment of capsules loaded with magnetic nanoparticles in a resting cell medium in the magnetic field of a permanent magnet. (B) Accumulation of capsules in the flow channel in the area of magnet location in a flowing cell medium. The images were obtained in a phase contrast mode.
1B). This leads to a strongly increased concentration of capsules in the region determined by the magnetic field gradient, which should promote the uptake of the capsules by the cells located nearby. The mechanism of ingestion of colloidal particles by cancer cells is discussed in a recent review paper.11 The more capsules are present locally, the more capsules should be ingested by the cells at this region. Indeed, Figure 2 demonstrates a strong difference in the accumulation and uptake of capsules by the cells located in the close vicinity and far from the permanent magnet. All cells which were examined for uptake statistics remained adherent at the bottom of the channel for the flow velocities chosen in our experiments, and only those capsules internalized by the cells were counted for the uptake statistics, as discussed in ref 5. For cells located far (11 mm) from the edge of the permanent magnet, no or very minor uptake of capsules was detected, as can be seen in a representative image (Figure 2A). For the cells located closer to the magnet edge (5 mm), a few internalized capsules per cell were generally detected (Figure 2B). In contrast, a strongly increased uptake was observed for the cells located just above the edge of the magnet (Figure 2C), reaching several tens of internalized capsules per cell. In addition, a large concentration of free capsules captured by the magnetic field gradient can be seen. Due to this local high concentration of capsules, the cells located
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Figure 2. Living breast cancer cells adherent at the bottom of the flow channel which are located (A) 11 mm away, (B) 5 mm away, and (C) just above the edge of the permanent magnet. Capsules taken up by the cells are recognizable by their luminescence. All pictures were obtained by overlay of phase contrast and luminescence images; the luminescence of capsules is determined by CdTe nanocrystals and is shown in pseudocolored red.
underneath ingest more capsules per cell than cells far away from the magnet. Figure 3 shows the statistics of the capsule uptake by cells located at three different positions (0, 5.5, and 11 mm away from the magnet edge) for three different flow velocities (5, 11, and 16 cm/s) of the cell medium. In all cases, a drastically increased uptake by the cells situated
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Figure 3. Statistics of capsules taken up by cells located at three different positions from the edge of the magnet (as indicated in frame A) for three different flow rates of the cell medium: (A) 5 cm/s, (B) 11 cm/s, and (C) 16 cm/s. Fifty individual cells have been counted in each case.
near the edge of the permanent magnet is observed, reaching several tens of internalized capsules per cell. Such high numbers of internalized capsules per cell might be explained by their partial squeezing, as previously reported.5 As expected, the uptake generally decreases for faster flow of the cell medium, although even for the fastest flow rate used in our experiments the magnetic field of the permanent magnet was strong enough to provide effective accumulation of capsules at desired places, promoting their uptake. Summarizing, our experiments demonstrate the possibility of specific localization of polymer capsules labeled by luminescent nanocrystals and functionalized with
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magnetic nanoparticles in a magnetic field gradient, followed by a drastically increased internalization of those capsules by breast cancer cells due to the high local concentration of capsules. This illustrates a way for magnetic delivery of polymer capsules loaded with pharmaceutical agents to pathogenic parts of a tissue, such as tumors, where the drugs could be released by opening of the capsule shells containing nanoparticles, for example, upon irradiation in the infrared.5,16 Additional modification of the capsule surface with specific receptor molecules (16) Skirtach, A. G.; Antipov, A. A.; Shchukin, D. G.; Sukhorukov, G. B. Langmuir 2004, 20, 6988.
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targeting only diseased cells could further increase the specificity of their uptake, reducing associated side-effects for the healthy cells. Relevant studies are under way, which ideally will lead to a multifunctional capsule-based carrier system with a strong impact on biotechnology. Acknowledgment. Financial support for this study was provided by the Volkswagen Foundation and by the Emmy Noether program of the German Research Foundation. LA0502286
