In vitro and in vivo transfection of primary phagocytes via microbubble-mediated intraphagosomal sonoporation

Abstract

The professional phagocytes, such as macrophages and dendritic cells, are the subject of numerous research efforts in immunology and cell biology. The use of primary phagocytes in these investigations however, are limited by their inherent resistance to transfection with DNA constructs. As a result, the use of phagocyte-like immortalized cell lines is widespread. While these cell lines are transfection permissive, they are generally regarded as poor biological substitutes for primary phagocytes. By exploiting the phagocytic machinery of primary phagocytes, we developed a non-viral method of DNA transfection of macrophages that employs intraphagosomal sonoporation mediated by internalized lipid-based microbubbles. This approach enables the transfection of primary phagocytes in vitro, with a modest, but reliable efficiency. Furthermore, this methodology was readily adapted to transfect murine peritoneal macrophages in vivo. This technology has immediate application to current research efforts and has potential for use in gene therapy and vaccination strategies.

Introduction

Professional phagocytes, such as macrophages and dendritic cells, function in a diverse array of physiologic, pathologic and immunologic roles in health and disease. Hence, the cellular biology of these lineages is a focus for numerous research areas. A major barrier to these research efforts is the high resistance of primary macrophages and dendritic cells to DNA transfection, together with the biological insufficiencies of transfection-permissive macrophage- and DC-like cell lines. Transfection resistance of the primary phagocytes is thought to be due to the innate ability of these cells to degrade foreign nucleic acids within the endolysosomal system (Burke et al., 2002). Methods that circumvent DNA degradation within these compartments, such as electroporation, also result in lower transfection efficiencies in phagocytes, indicating that there are additional factors contributing to this resistance (Takahashi et al., 1992, Burke et al., 2002). While some success has been achieved using viral methods of gene transfer, these are typically limited by the size of foreign DNA and their suitability and selectivity for in vivo applications. In addition, the construction of recombinant viruses, particularly for a large number of constructs, is often time-prohibitive. Hence non-viral methods of gene delivery to primary phagocytes, although typically yielding lower transfection efficiencies and cell viabilities, are still preferred in certain scenarios. In particular, non-viral transfection of ex vivo and in vivo macrophages and dendritic cells has generated interest for the potential to use these cells as targets or vehicles in gene therapy (Burke et al., 2002). Electroporation of macrophages can be achieved using systems such as the commercially available Nucleofector™ kit marketed by Lonza. This method, although popular for macrophage-like cell lines, is limited to in vitro applications and is associated with low viability and altered cellular morphology (Van De Parre et al., 2005). Due to the limitations of current methodologies for transfection of primary bone marrow derived macrophages (BMMØs), we sought to develop a novel, non-viral, phagocyte-specific method of targeted gene delivery. The method we have developed utilizes intraphagosomal sonoporation mediated by internalized lipid-based microbubbles.

Originally developed as contrast agents for use in diagnostic ultrasonography, gas-filled encapsulated microbubbles (herein referred to as microbubbles) have recently received attention for their potential use as gene delivery vectors in genetic therapy (Newman and Bettinger, 2007). Microbubbles are typically 1–3 μm in diameter and consist of heavy-gas cores (perfluorocarbons or sulphur hexafluoride) with stabilizing shells composed of lipid, protein or synthetic polymer (Lindner, 2004). In addition to the acoustic properties, which allow microbubbles to be used as contrast agents, inertial cavitation of microbubbles following insonation results in a large, localized release of mechanical energy (Sundaram et al., 2003, Schlicher et al., 2006). This energy can be released in the form of hydrodynamic shock waves with induced pressures of 4 mPa, microjets with a velocity of 5.5 m s^− 1, and pressures of 15 kPa. Both shockwaves and microjets are capable of transiently breaching biological membranes which have an estimated critical pressure threshold of 3 kPa (Prentice et al., 2005, Zhao et al., 2008). This phenomenon has been termed “microbubble-mediated sonoporation”, and has been shown to produce pores up to 100 nm in diameter with half lives of a few seconds (Newman and Bettinger, 2007). Introduction of microbubble-associated plasmid DNA into non-phagocytic eukaryotic cells through microbubble-induced sonoporation has been demonstrated both in vitro and in vivo (Newman and Bettinger, 2007).

We hypothesized that we could utilize the phagocytic machinery of primary phagocytes to facilitate their transfection through intraphagosomal sonoporation by the cavitation of internalized microbubbles. Furthermore, we reasoned that sonoporation of perinuclear phagosomes would yield a higher efficiency of transfection due to their proximity to the nucleus and be better tolerated than sonoporation of the plasma membrane. To specifically target phagocytes and to stimulate phagocytic uptake, we developed a cationic-, biotinylated-, lipid-coated microbubble that could be opsonized with an anti-biotin immunoglobulin (IgG) antibody and to which plasmid DNA could be electrostatically bound to the cationic shell. Following phagocytosis, cavitation of the microbubble could be induced by external insonation, which in some cases could result in transfection of the primary phagocyte. This method of transfection we term “microbubble-mediated intra-phagosomal sonoporation (MIPS)”. Although in vitro transfection efficiencies using this novel methodology were modest, transfected phagocytes were morphologically indistinguishable from untransfected controls. Moreover, using this approach we achieved the in vivo transfection of murine peritoneal macrophages. This not only demonstrated the potential of this and similar reagents in future genetic therapies targeting phagocytes, but more immediately provided a research tool that permits transfection and expression of DNA constructs within phagocytes of animal models.