Microinjection for the ex Vivo Modification of Cells with Artificial Organelles Peter Tiefenboeck, Jong Ah Kim, Ferdinand Trunk, Tamara Eicher, Erica Russo, Alvaro Teijeira, Cornelia Halin, and Jean-Christophe Leroux* Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zürich, 8093 Zürich, Switzerland S Supporting Information *
ABSTRACT: Microinjection is extensively used across fields to deliver material intracellularly. Here we address the fundamental aspects of introducing exogenous organelles into cells to endow them with artificial functions. Nanocarriers encapsulating biologically active cargo or extreme intraluminal pH were injected directly into the cytosol of cells, where they bypassed subcellular processing pathways and remained intact for several days. Nanocarriers’ size was found to dictate their intracellular distribution pattern upon injection, with larger vesicles adopting polarized agglomerated distributions and smaller colloids spreading evenly in the cytosol. This in turn determined the symmetry or asymmetry of their dilution following cell division, ultimately affecting the intracellular dose at a cell population level. As an example of microinjection’s applicability, a cell type relevant for cell-based therapies (dendritic cells) was injected with vesicles, and its migratory properties were studied in a co-culture system mimicking lymphatic capillaries. KEYWORDS: microinjection, nanocarriers, liposomes, bioreactors, cell division, cell migration, cell-based therapy
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efficiency, but controlling the amount of nanocarrier internalized during co-incubation can be challenging since it depends on the cells’ often heterogeneous endocytic activity. Indeed some cell types that are clinically relevant for cell-based therapies have limited endocytic capacity (e.g., immune cells, stem cells). In addition, unless effective strategies are applied to enable escape into the cytosol,16,17 the endocytosed material can be degraded downstream in the endolysosomal compartment, suppressing its biological activity.4,18 Membranedisrupting techniques have thus emerged as alternative methods to deliver intracellularly various kinds of nanocarriers to virtually any cell type, although not without their own limitations compared to co-incubation.19,20 Based on either the permeabilization or the direct penetration of the plasma membrane, these methods introduce material directly into the cytoplasm (or nucleus), bypassing not only the physical barrier that the membrane imposes to hydrophilic molecules but also degradative compartments.21−23 Electroporation,24 cell-squeezing,25 nanoneedles,26 and microinjection27 are among the most exploited ones. However, depending on their mechanism of action, some of these techniques might impose size restrictions for the material dispensed to cells25 or imply some degree of cytotoxicity,24 which could constitute a limiting aspect in view of the therapeutic applications of the modified cells.28−30
ell-based therapies propose to exploit migratory cells as vehicles to transport therapeutic agents and deliver them at sites of inflammation or malignant growth to which they get recruited.1 Most often macrophages, lymphocytes, and dendritic cells (DCs) are used, but recently also microorganisms have been proposed.2 Regardless of the carrier cell used, the transported cargo needs to be first encapsulated into a nanocarrier (e.g., nanoparticle, vesicle) to preserve its stability3 and to protect the cell itself from the drug’s effects.4 Ex vivo-manipulated macrophages and lymphocytes have been used to deliver nanocarriers loaded with chemotherapeutics,5,6 antiretroviral drugs,7 proteins or enzymes,8,9 and imaging contrast agents.10 In the context of cancer immunotherapy, DCs have been endowed ex vivo with encapsulated adjuvants and cancer antigens to develop cell-based vaccines.11,12 Magnetotactic bacteria that migrate toward hypoxic regions have been used in mice to deliver liposomes bearing an antitumor drug.13 Besides boosting the drugs’ efficacy by increasing the dose achieved on-site, carrier cells can minimize side effects associated with systemic exposure.14 Furthermore, transporting degradation-prone cargo (e.g., therapeutic proteins, nucleic acids) inside cellular vehicles can increase their half-life and preserve their function.4 Notably, the method with which the nanocarriers are introduced into the recipient cells has a deep effect on the cargo’s stability. Their co-incubation is an extensively used approach, relying on the cell’s endocytic mechanisms to internalize the nanocarriers.15 This is a convenient method in terms of ease of handling and time © 2017 American Chemical Society
Received: February 27, 2017 Accepted: August 4, 2017 Published: August 4, 2017 7758
DOI: 10.1021/acsnano.7b01404 ACS Nano 2017, 11, 7758−7769
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Figure 1. Microinjection of liposomal formulations into cells. (a) Schematic representation of liposomes’ microinjection into a cell. The microinjector tip pierces the cell’s plasma membrane and injects the liposomes directly into the cytoplasm. Liposomes can be fluorescently labeled (red coronas) or loaded with different cargoes (blue dots in the liposomes’ cores), and a co-injection marker can be included in the suspension to identify the injected cells. (b) Cell viability measured by propidium iodide exclusion 0, 4, 24, and 48 h after injection of the marker alone (Cy5), DPPC-PEG LUVs, DPPC LUVs, DOPC-PEG LUVs, DOPC LUVs, and SA LUVs. Values were normalized for total injected cells at time 0 (mean + SD, n = 285−355 in 3−4 independent experiments). ***p-value
