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Multi-compartment Artificial Organelles Conducting Enzymatic Cascade Reactions inside Cells Maria Godoy-Gallardo, Cedric Labay, Vasileios Trikalitis, Paul Kempen, Jannik B Larsen, Thomas Lars Andresen, and Leticia Hosta-Rigau ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b16275 • Publication Date (Web): 24 Jan 2017 Downloaded from http://pubs.acs.org on January 25, 2017
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ACS Applied Materials & Interfaces
Multi-compartment Artificial Organelles Conducting Enzymatic Cascade Reactions inside Cells Maria Godoy-Gallardo, Cédric Labay, Vasileios D. Trikalitis, Paul J. Kempen, Jannik B. Larsen, Thomas L. Andresen and Leticia Hosta-Rigau*
Department of Micro- and Nanotechnology, Centre for Nanomedicine and Theranostics, DTU Nanotech, Technical University of Denmark, Building 423, 2800, Lyngby, Denmark E-mail:
[email protected] KEYWORDS. Capsosomes, enzymatic cascade reactions, fluorescent gold nanoclusters, intracellular microreactors, liposomes
ABSTRACT. Cell organelles are subcellular structures entrapping a set of enzymes to achieve a specific functionality. The incorporation of artificial organelles into cells is a novel medical paradigm which might contribute to the treatment of various cell disorders by replacing malfunctioning organelles. In particular, artificial organelles are expected to be a powerful solution in the context of enzyme replacement therapy since enzymatic malfunction is the primary cause of organelle dysfunction. Although several attempts have been made to encapsulate enzymes within a carrier vehicle, only few intracellularly active artificial organelles have been reported to date and they all consist of single-compartment carriers. However, it is
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noted that biological organelles consist of multicompartment architectures where enzymatic reactions are executed within distinct sub-compartments. Compartmentalization allows for multiple processes to take place in close vicinity and in a parallel manner without the risk of interference or degradation. Here, we report on a sub-compartmentalized and intracellularly active carrier; a crucial step for advancing artificial organelles. In particular, we develop and characterize a novel capsosome system, which consists of multiple liposomes and fluorescent gold nanoclusters embedded within a polymer carrier capsule. We subsequently demonstrate that encapsulated enzymes preserve their activity intracellularly, allowing for controlled enzymatic cascade reaction within a host cell.
1. INTRODUCTION Since malfunction at a cellular level is the primary cause of most diseases, the creation of micro/nanoreactors that can act intracellularly to compensate for lost or decreased cellular function will add a novel paradigm for medical therapy. Those micro/nanoreactors, known as artificial organelles, have the potential to yield a therapeutic effect in dysfunctional cells and/or extend biosynthetic pathways in living cells.1,2 One particular very promising application of such micro/nanoreactors is in the field of enzyme replacement therapy.1 Enzyme malfunction is the primary cause of organelle dysfunction and, current strategies for the delivery of enzymes, are facing major difficulties in clinical trials since enzymes do not possess sufficient in vivo stability, are not easily taken up by cells or are intracellularly trafficked in vivo.3,4 The creation of artificial organelles with encapsulated enzymes, which are protected from degradation by cellular contents allowing them to perform
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their catalytic activity for an extended period of time, is expected to overcome the limitations of current enzyme-therapy. For the design of advanced artificial organelles, certain design criteria need to be fulfilled. A major feature of cell organelles is that they operate by conducting multiple (cascade) reactions executed within confined, distinct sub-compartments. Multi-compartmentalization is crucial for their survival, since the spatial separation of both biomolecules and processes provided by biological organelles allows to spatially separate reaction pathways and/or incompatible components.5 Within cellular organelles, multiple metabolic processes can take place without interfering with each other or being degraded by other factors (e.g., the Golgi apparatus or the mitochondria, in which several enzymes for the citric acid cycle are located in the intermembrane space).6,7 However, the construction of multi-compartment architectures has already proven very challenging and only a few systems have succeeded in dividing their internal space in a controlled manner. Liposomes-in-liposomes,8,9 polymersomes-in-polymersomes5,10 and capsosomes,11,12 are the most advanced and well-studied carriers containing multiple compartments reported to date. In contrast to the other multi-compartment architectures, capsosomes offer unique properties arising from the combination of two inherently different building blocks. By combining liposomes and polymer capsules, capsosomes exploit the benefits of both systems while minimizing some of their drawbacks. The liposome subunits have the ability to protect fragile contents such as enzymes, preventing them from misfolding or denaturation,13 however, liposomes have some limitations such as low in vivo stability, lack of control over their degradability,14,15 and are largely impermeable to their surroundings.16 The polymer carrier shell prepared by the layer-by-layer (LbL) technique, which is based on the deposition of interacting polymers onto sacrificial core templates,17 balances the liposomes
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drawbacks by supplying the structural integrity and protecting the liposomes from degradation.18 Additionally, the semipermeable nature of the polymer membrane allows for free permeation of substrate in and out the microreactor; a crucial factor to conduct continuous (enzymatic) reactions. Once the multi-compartment architecture has been achieved, the next step for an artificial organelle to support its host cell is to be internalized. Similar to our previous report,19 we evaluated the capsosomes uptake by a macrophage cell line in an in vitro setup and demonstrated that capsosomes can be successfully integrated within macrophages. Macrophages represent the first line of defence against intruding pathogens and can be found to be dysfunctional in diseases such as atherosclerosis,20 cancer21 or autoimmune disorders.22 In addition to the incorporation of biomimetic features, the synthetic nature of artificial organelles allows for controlled augmentation of specific properties, hereby adding novel features not inherent to the host cell. To expand the functionality of our artificial organelle we incorporate gold nanoclusters (Au NCs) allowing us to detect and track our compartment carrier within the host cell.19 We chose to incorporate sub-nanometer sized (
