Intracellular Release DOI: 10.1002/ange.201206696 Light-Addressable Capsules as Caged Compound Matrix for Controlled Triggering of Cytosolic Reactions** Markus Ochs, Susana Carregal-Romero, Joanna Rejman, Kevin Braeckmans, Stefaan C. De Smedt, and Wolfgang J. Parak* Layer-by-layer assembly was introduced almost two decades ago as a versatile technique for the construction of thin multiple-layer films composed out of polyelectrolytes. [1, 2] Shortly after, the concept was extended from planar to spherical geometry, resulting in polyelectrolyte multilayer capsules. [3–5] The semipermeable wall of the capsules (with a thickness of a few nanometers) [6, 7] and the cavity can be further loaded with inorganic colloidal nanoparticles (NPs) made of different materials and with multiple cargos, [8–10] respectively (Figure 1). The resulting multifunctional capsules are well-suited for in vitro delivery of cargo inside cells. [11, 12] This concept has been highlighted in several recent reviews. [13–15] Meanwhile technology has advanced to a point at which these capsules could be a helpful tool for controlled multifunctional in vitro delivery. Nowadays the cavity of capsules can be loaded with a large variety of cargo. While large molecules such as proteins will be readily kept inside the cavity, small molecules need to be either linked to macro- molecules such as dextran, [16] or be embedded inside micelles. [17] The micelle approach even allows for encapsula- tion of small hydrophobic molecules. The materials forming the polyelectrolyte wall can be chosen such that capsules internalized by cells are not degraded and preserve their cargo over weeks. Leakage of the cargo molecule is reduced and controlled release of cargo upon external stimuli can be performed. A large number of capsules can be taken up by cells in vitro without causing acute cytotoxicity, even for capsules with large sizes of around 5 mm. [18–20] It is conceivable that if cells are exposed simultaneously to different types of capsules with similar layer composition, they will internalize them with a statistical distribution. To demonstrate this we exposed cells to a mixture of four types of capsules loaded with different fluorescently labeled dextrans emitting blue, green, red, or near-infrared light (Figure 2A) corresponding to Cascade Blue, fluorescein isothiocyanate, AlexaFluor594, and Dy647, respectively. HeLa cells were incubated with amounts of capsules equivalent to two, four, or six capsules of each color per cell for four hours. Figure 2B presents the number of capsules of each color internalized per cell. When six capsules of each color (i.e. 24 capsules in total) were added per cell, 50 % of the cells had internalized at least one capsule of each color. Contrary, when only two capsules per cell of the four kinds were added the percentage dropped to less than 20 %. These findings demonstrate the feasibility to simultaneously load cells with a variety of encapsulated cargos. The loading of cells can be specifically directed by incorporating magnetic NPs in the wall of the capsules. This is possible since magnetic field gradients, which are created by positioning a magnet in a flow channel system, trap the capsules close to the magnet. [21] This method can ultimately be used to achieve specific capsule distribution patterns in the cell culture or in vivo for certain applications. Figure 2C,D shows the results of an experiment demonstrating the targeted deposition of differently colored capsules in a sub- millimeter pattern. A magnet with an edge length of 5 mm (ca. 1,3 T) was modified with two iron slips on top (width ca. 800 mm) that apply the magnetic field in the shape of two stripes underneath a flow channel, which simulates blood Figure 1. A) Schematic representation of a capsule with walls produced via layer-by-layer assembly (gray). The cavity of the capsule is loaded with a cargo (blue). The wall of the capsule contains magnetic (black) and plasmonic (red) NPs. B) A representative transmission electron microscopy (TEM) image of a capsule with a large amount of magnetic Fe 2 O 3 and plasmonic Au NPs in its walls. The scale bar corresponds to 1 mm. [*] M. Ochs, [+] Dr. S. Carregal-Romero, [+] Prof. W. J. Parak Fachbereich Physik and WZWM, Philipps Universität Marburg Renthof 7, 35037 Marburg (Germany) E-mail: wolfgang.parak@physik.uni-marburg.de Dr. S. Carregal-Romero [+] Bionand. Severo Ochoa 35, 29590 Mµlaga (Spain) Dr. J. Rejman, Prof. K. Braeckmans, Prof. S. C. De Smedt Laboratory of General Biochemistry and Physical Pharmacy Ghent University Harelbekestraat 72, Ghent (Belgium) Prof. K. Braeckmans Center for Nano and Biophotonics, Ghent University Harelbekestraat 72, Ghent (Belgium) [ + ] These authors contributed equally to this work. [**] This work was supported by BMBF/ERANET (project Nanosyn) and the DFG (project PA794/11.1). We acknowledge technical discus- sions with Drs. Rafael Fernandez Chacón, Loretta del Mercato, Arnold Grünweller, Roland Hartmann, Pilar Riveral Gil and Gleb B. Sukhorukov. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201206696. 723 Angew. Chem. 2013, 125, 723 –727  2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim