Application of pulsed-magnetic field enhances non-viral gene delivery in primary cells from different origins

Abstract

Primary cell lines are more difficult to transfect when compared to immortalized/transformed cell lines, and hence new techniques are required to enhance the transfection efficiency in these cells. We isolated and established primary cultures of synoviocytes, chondrocytes, osteoblasts, melanocytes, macrophages, lung fibroblasts, and embryonic fibroblasts. These cells differed in several properties, and hence were a good representative sample of cells that would be targeted for expression and delivery of therapeutic genes in vivo. The efficiency of gene delivery in all these cells was enhanced using polyethylenimine-coated polyMAG magnetic nanoparticles, and the rates (17–84.2%) surpassed those previously achieved using other methods, especially in cells that are difficult to transfect. The application of permanent and pulsating magnetic fields significantly enhanced the transfection efficiencies in synoviocytes, chondrocytes, osteoblasts, melanocytes and lung fibroblasts, within 5 min of exposure to these magnetic fields. This is an added advantage for future in vivo applications, where rapid gene delivery is required before systemic clearance or filtration of the gene vectors occurs.

Introduction

Primary cell cultures isolated from different organs have been successfully cultivated in culture medium. The use of these cells to express therapeutic genes would be a step forward in providing safer therapeutic approaches with fewer side effects, which are usually associated with systemic administration of drugs. In general, primary cells are more difficult to transfect when compared to immortalized/transformed cell lines [1], [2] and hence there is need to develop new techniques to enhance transfection efficiency, since only these cells (as autologous carriers) would be most suited for delivery and expression of therapeutic genes in the respective organs. Depending on the organ of origin, primary cells differ in properties, which should be considered in elucidating their full potential in expression and delivery of therapeutic genes.

Chondrocytes are the native cells of the articular cartilage, and therefore are logical targets for transfer and expression of therapeutic genes for treatment of incurable joint diseases. However, because of the dense cartilage extracellular matrix that surrounds these cells, they are unavailable for genetic modification by direct intra-articular injection of most recombinant vectors [3], [4], [5], [6], [7]. Therefore, in many cases, successful cartilage repair would require administration of supplemental cells transduced with therapeutic genes. Synovial cells which consist of a large cell population that cover a significant surface area of the joint cavity would be promising alternative to chondrocytes in treatment of joint diseases. Osteoblasts isolated from bone marrow stromal stem cells and osteoprogenitor cells are easily cultivated in vitro, and successful ex vivo gene transfer in osteoblasts would be an advantageous strategy for delivering the signaling molecules that would stimulate migration and proliferation, promoting differentiation [8], [9], [10], [11], [12], [13], [14], and eventually enhancing mineralization of the bone.

Primary chondrocytes and osteoblasts are receptive to transduction using the more common viral vectors, however, the use of these vectors are still limited by safety concerns, such as virus-induced inflammatory response, which can cause a myriad of side effects ranging from mild eodema to multi-system organ failure. The immune system's enhanced response to the now recognized virus makes it difficult to administer gene therapy repeatedly. At a more cellular level, transfection with some viruses have been shown to cause significant changes in the cell surface markers of transfected cells, decreasing the possibility of reusing the same population of cells for a second round of gene delivery [15], [16], [17], [18], [19], [20]. Non-viral gene vectors on the other hand have the advantages of low cost, easy manufacture, low immunogenicity and offer the possibility of repeating dosing [21], [22], however, the transfection efficiencies achieved are much lower. Transfection rates of 31.3%, 30.3% and 8.3%, were achieved in chondrocytes transfected with polyamidoamine, linear polyethylenimine (PEI) and branched PEI, respectively [23], while in osteoblasts, the maximum transfection rates achieved with minimum cytotoxicity, were 12.3% and 8.3%, when lipofectamine and PEI were used, respectively [14]. Chitosan nanoparticles resulted in transfection rates of up to 50% in chondrocytes [24], [25], however, these nanoparticles were poor gene delivery vectors in synovial cells [25]. Some increase in transfection efficiency in chondrocytes was achieved by the addition of hyaluronidase to FUGENE [26], [27], [28] or by use of a detergent (lysolecithin) to permiabilize the cells, in transfection using poly-l-lysine liposomes covalently linked to a receptor ligand, transferrin [29]. However, simpler, more efficient and direct gene delivery techniques for these cells are still required.

Macrophages have a crucial function in a variety of biological processes and pathologies which renders them as important targets for gene therapeutic interventions, however, gene transfection of macrophages, has proven difficult [30], [31], [32]. Several non-viral gene transfer methods have been developed to improve transfection of these cells, however, these have been associated with low-transfection efficiency [31], [32]. In addition to the above cells, enhancing non-viral gene delivery to primary melanocytes would be a step forward in treatment of melanomas, which is usually accompanied by resistance to therapy [33].

One way to bridge the gap in effectiveness between the viral and non-viral systems is to optimize the non-viral system to its full potential. Rapid and efficient gene delivery in primary cell lines is paramount in vivo, where rapid systemic clearance and filtration of gene vectors occurs. We recently showed that PEI-coated magnetic nanoparticles were very efficient non-viral gene delivery vectors in established cell lines, within 5 min of exposure to magnetic field [34]. The PEI-coated PolyMAG magnetic nanoparticles [35], [36], [37] condense DNA by electrostatic interaction, which protects the DNA from enzymatic degradation [21], [22], [34], [38], [39], [40]. The uptake of DNA/PolyMAG complexes is by unspecific endocytosis [41] and presence of a magnetic field has been shown to results in dramatic increase in transfection efficiency [34], [41], [42], [43], [44], [45].

To determine the efficiency of PolyMAG nanoparticles in gene delivery in primary cells, we isolated primary cells from three different animal models. Chondrocytes, synoviocytes and osteoblasts were isolated from sheep, which have become a favored model for orthopedic research. This is due to their similarity with humans in weight, bone and joint structure, and bone regeneration. Additionally, availability, easy of handling and housing, animal costs, and acceptance by society, make sheep ideal research model [46], [47], [48], [49]. Melanocytes were isolated from cutaneous melanoma of gray horses. Gray horses spontaneously develop metastatic melanomas that resemble human disease, and this is accompanied with metastasis to other organs [50], [51]. Previous studies had shown that intratumoral injection of interleukin-18 and interleukin-12 DNAs showed some inhibitory effect on melanoma growth in gray horses [50]. In a follow up study we will establish if intratumoral injections of primary melanocytes transfected with interleukin−18 and interleukin−12 DNAs would result to tumor regression. Enhancement of gene delivery in primary equine melanocytes is therefore highly relevant for these future studies. Lung fibroblasts, peritoneum macrophages and embryonic fibroblasts were isolated from mice, the animal model used in most in vivo studies. Enhancing transfection in primary cells from this model would have several advantages in future studies that will use mouse model or cells derived from it. Our study therefore constituted a good representative sample of primary cells that could be targeted for expression of therapeutic genes in different disease conditions. The effect of static (permanent) and pulsating magnetic fields on the transfection efficiencies was determined after different exposure durations and compared to that of other non-viral gene vectors.