Internationale Ausgabe: DOI: 10.1002/anie.201606960 Soft Matter Deutsche Ausgabe: DOI: 10.1002/ange.201606960 One-Step Microfluidic Fabrication of Polyelectrolyte Microcapsules in Aqueous Conditions for Protein Release Liyuan Zhang + , Li-Heng Cai + , PhilippS. Lienemann, Torsten Rossow, Ingmar Polenz, Queralt Vallmajo-Martin, Martin Ehrbar, Hui Na, David J. Mooney, and David A. Weitz* Abstract: We report a microfluidic approach for one-step fabrication of polyelectrolyte microcapsules in aqueous con- ditions. Using two immiscible aqueous polymer solutions, we generate transient water-in-water-in-water double emulsion droplets and use them as templates to fabricate polyelectrolyte microcapsules. The capsule shell is formed by the complex- ation of oppositely charged polyelectrolytes at the immiscible interface. We find that attractive electrostatic interactions can significantly prolong the release of charged molecules. More- over, we demonstrate the application of these microcapsules in encapsulation and release of proteins without impairing their biological activities. Our platform should benefit a wide range of applications that require encapsulation and sustained release of molecules in aqueous environments. Polyelectrolyte (PE) microcapsules are aqueous droplets surrounded by a shell that is solidified owing to the electro- static attraction between oppositely charged polymers rather than by crosslinking polymers using chemical reactions. [1] As such, they are widely used for encapsulation and release of chemically sensitive reagents in the pharmaceutical and food industries. [2] The classical way to fabricate PE microcapsules, however, requires solid particles as templates; this method uses layer-by-layer assembly to alternatively deposit oppo- sitely charged polyelectrolytes onto the surface of a micro- meter-scale particle and subsequently decomposes the tem- plate material into individual molecules that can pass through the shell. [3] The process of decomposition, however, often impairs the shell integrity and thus results in non-sustained release of the encapsulated contents. [4] Alternatively, PE microcapsules can be fabricated using emulsions, rather than solid particles, as templates. For example, using water-in-oil emulsions generated by microfluidic techniques, PE micro- capsules have been successfully fabricated based on the complexation of oppositely charged polyelectrolytes at the water/oil interface. [5] Despite the promise of this emulsion- based technique, its use is severely limited because of the requirements of using organic solvents. Avoiding these solvents would greatly advance the application of PE micro- capsules in encapsulating chemically sensitive biomolecules such as proteins; however, there remains an unmet need for the development of a simple method that enables the fabrication of PE microcapsules in biologically friendly aqueous conditions for protein release. Herein, we report a one-step microfluidic fabrication of PE microcapsules in aqueous conditions. Unlike traditional methods that rely on organic–aqueous systems, we use two aqueous polymer solutions with a relatively small interfacial tension, yet large enough to generate transient water-in- water-in-water (W/W/W) double emulsion drops. [6] We use these transient W/W/W drops as templates to fabricate PE microcapsules based on the interfacial complexation of oppositely charged polyelectrolytes. Remarkably, the shell thickness of PE microcapsules is nearly independent of the concentration and molecular weight (MW) of the polyelec- trolytes. We quantify the release profile of the PE micro- capsules for both neutral and charged molecules. Interest- ingly, the release of negatively charged molecules is signifi- cantly prolonged because of attractive electrostatic interac- tions. Finally, we demonstrate the application of PE micro- capsules in encapsulation and release of platelet-derived growth factor-BB (PDGF-BB) without impairing its biolog- ical activities. We use a glass capillary microfluidic device to generate transient W/W/W emulsion drops that serve as templates for the formation of microcapsules (Supporting Information, Materials and Methods). [7] The device consists of two tapered cylindrical capillaries that are inserted into opposite ends of a square capillary, as illustrated by Figure 1a. We use the left capillary to inject the inner phase consisting of 15 wt % dextran and 0.5 wt % poly(diallyldimethyl ammonium chlo- ride) (PDDA), a positively charged polyelectrolyte (PE+). The middle phase, 17 wt% polyethylene glycol (PEG), is injected from the left through the interstices between the left cylindrical and the square capillaries. The outer phase is injected from the right interstices; it is essentially the same [*] Dr. L. Zhang, [+] Dr. L.-H. Cai, [+] Dr. P.S. Lienemann, Dr. T. Rossow, Dr. I. Polenz, Dr. D. J. Mooney, Dr. D. A. Weitz John A. Paulson School of Engineering and Applied Sciences, Harvard University Cambridge, MA 02138 (USA) E-mail: weitz@seas.harvard.edu Dr. P.S. Lienemann, Dr. T. Rossow, Dr. D.J. Mooney Wyss Institute for Biologically Inspired Engineering Harvard University Boston, MA 02115 (USA) Q. Vallmajo-Martin, Dr. M. Ehrbar Department of Obstetrics, University Hospital Zurich University of Zurich Schmelzbergstr, 12, 8091 Zurich (Switzerland) Dr. H. Na Alan G MacDiarmid Institute, College of Chemistry, Jilin University Changchun, 130012 (China) [ + ] These authors contributed equally to this work. Supporting information (including all experimental details) and the ORCID identification number(s) for the author(s) of this article can be found under: http://dx.doi.org/10.1002/anie.201606960. A ngewandte Chemi e Zuschriften 1 Angew. Chem. 2016, 128,1–6  2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim These are not the final page numbers! Ü Ü