4785 © 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com Intracellular Implantation of Enzymes in Hollow Silica Nanospheres for Protein Therapy: Cascade System of Superoxide Dismutase and Catalase Feng-Peng Chang, Yi-Ping Chen, and Chung-Yuan Mou* 1. Introduction In biological cells, a great variety of enzymatic catalysis is responsible for energy conversion, synthesis, and cellular defense to maintain physiological functionality. To tightly regulate multiple reactive species in the crowded cellular environment, compartmentalization is a dominant feature in eukaryotic cells. This is to keep enzymes and biomole- cules together to reach a higher concentration and to lead DOI: 10.1002/smll.201401559 An approach for enzyme therapeutics is elaborated with cell-implanted nanoreactors that are based on multiple enzymes encapsulated in hollow silica nanospheres (HSNs). The synthesis of HSNs is carried out by silica sol–gel templating of water-in-oil microemulsions so that polyethyleneimine (PEI) modified enzymes in aqueous phase are encapsulated inside the HSNs. PEI-grafted superoxide dismutase (PEI-SOD) and catalase (PEI-CAT) encapsulated in HSNs are prepared with quantitative control of the enzyme loadings. Excellent activities of superoxide dismutation by PEI-SOD@HSN are found and transformation of H 2 O 2 to water by PEI-CAT@ HSN. When PEI-SOD and PEI-CAT are co-encapsulated, cascade transformation of superoxide through hydrogen peroxide to water was facile. Substantial fractions of HSNs exhibit endosome escape to cytosol after their delivery to cells. The production of downstream reactive oxygen species (ROS) and COX-2/p-p38 expression show that co-encapsulated SOD/CAT inside the HSNs renders the highest cell protection against the toxicant N,N ′-dimethyl-4,4′-bipyridinium dichloride (paraquat). The rapid cell uptake and strong detoxification effect on superoxide radicals by the SOD/CAT- encapsulated hollow mesoporous silica nanoparticles demonstrate the general concept of implanting catalytic nanoreactors in biological cells with designed functions. Hollow Spheres F.-P. Chang, Prof. C.-Y. Mou Department of Chemistry National Taiwan University Taipei 106, Taiwan E-mail: cymou@ntu.edu.tw Dr. Y.-P. Chen Research Center for Applied Sciences Academia Sinica Taipei 115, Taiwan to orderly control of complex cascade reactions. Cascade enzymatic reactions of the glyoxylate cycle, Calvin cycle, and Krebs cycle in an organelle enclosure are examples of cata- lyzed reactions by confined enzymes. Inspired by the compartmentalized structure in living cells, building hollow porous nanostructured materials to encapsulate multiple enzymes would be a very promising approach in biomedicine for enhancing, repairing, or adding new functionalities to cells. [1] Such strategy of synthetic organelles implanted into cells is still at its nascent stage of development. [2] Tandem enzyme reactions are mostly dem- onstrated in test tubes using liposomes, [3] polymer-based materials, [4–11] or gelatin microcapsules. [12] The challenges of existing materials are overcoming poor stability, their micrometer-size range, their slow cell uptake, and low perme- ability toward small molecules that hampers their function as organelle-mimics in cells. Recently, we reported a microemulsion-template method [9] for synthesizing hollow silica nanospheres (HSNs), with large interior spaces and porous silica shells, suitable small 2014, 10, No. 22, 4785–4795