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[摘要]：Technologies currently available for the controlled release of protein therapeutics involve either continuous or tissue-specific discharge from implants or engineered extracellular matrix mimetics. For some therapeutic applications the trigger-controlled release of protein cargo from a synthetic implant could be highly desirable. We have designed the CellEase technology, a two-component system consisting of cellulose sulfate (CS) poly-diallyldimethyl ammonium chloride (pDADMAC) capsules harboring mammalian sensor cells transgenic for trigger-inducible expression of an engineered secreted mammalian cellulase (SecCell). SecCell is a Bacillus subtilis-derived (1 -4)-beta-glucanase, which was modified by replacing the N-terminal part of the bacterial enzyme with a murine IgK-chain V-12-C region-derived secretion signal. SecCell was engineered for doxycycline- or erythromycin-inducible expression to enable trigger-controlled secretion by mammalian cells. Detailed characterization of SecCell showed that it was glycosylated and efficiently secreted by a variety of mammalian sensor cells such that it could internally rupture CS-pDADMAC capsules within which the cells had been encapsulated. When SecCell was inducibly expressed in sender cells, that were co-encapsulated with producer cell lines expressing therapeutic proteins, the removal of relevant inducer molecules enabled the time-dependent discharge of these therapeutic proteins, the kinetics of which could be modified by varying the concentration of inducer molecules or the amount of encapsulated sender cells. SecCell's capacity to rupture CS-pDADMAC capsules exclusively internally also enabled the independent trigger-induced release of different proteins from two capsule populations harboring different inducible SecCell sensor cells. CellEase-based protein release was demonstrated in vivo using capsules implanted intraperitoneally into mice that enabled the doxycycline-controlled release of a model glycoprotein and accumulation in the bloodstream of treated animals. Trigger-induced breakdown of tissue-compatible implants which provide a timely controlled release of biologics may foster novel opportunities in human therapy. (c) 2008 Elsevier B.V. All rights reserved. |
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