mRNA transfection of cervical carcinoma and mesenchymal stem cells mediated by cationic carriers Joanna Rejman 1 , Geertrui Tavernier 1 , Neda Bavarsad, Joseph Demeester, Stefaan C. De Smedt ⁎ Ghent University, Laboratory of General Biochemistry and Physical Pharmacy, Ghent Research Group on Nanomedicines, Harelbekestraat 72, 9000 Ghent, Belgium abstract article info Article history: Received 30 April 2010 Accepted 29 July 2010 Available online 12 August 2010 Keywords: Delivery mRNA Cationic carriers Lipoplexes Polyplexes Messenger RNA encoding luciferase (mLUC) was complexed to the cationic lipids Lipofectamine or DOTAP/ DOPE, and to the cationic polymer linear poly(ethyleneimine) (linPEI). The complexes were incubated with HeLa cells and luciferase expression was assessed. The type of non-viral carrier used determined the extent and duration of protein expression. Maximal duration of mRNA expression was about 9 days for Lipofectamine complexes, i.e. not very much shorter than with pDNA polyplexes. Interestingly, luciferase activity was already detected 30 min after adding the mRNA complexes to the cells, independent on the type of carrier. We also assessed the proportion of cells that become transfected by means of transfection with an mRNA encoding GFP. For both cationic lipids transfection with mRNA yielded a substantially larger fraction of transfected cells (more than 80%) than transfection with pDNA (40%). In addition we tested the carriers for their ability to mediate delivery of mRNA encoding CXCR4 into mesenchymal stem cells. The fraction of CXCR4-positive cells obtained with the mRNA–cationic lipid complexes was around 80%, as compared to 40% for the linPEI polyplexes. Our results demonstrate that the advantage of the use of mRNA over that of pDNA may under certain conditions outweigh the disadvantage of the somewhat shorter expression period. © 2010 Elsevier B.V. All rights reserved. 1. Introduction It is well known that cationic carriers can condense plasmid DNA into positively charged complexes. Such complexes can interact with the plasma membrane and be taken up by cells, most commonly by endocytosis [1–3]. To ensure transfection, the complexes have to escape the endosomal compartment and release pDNA into the cytosol. In the last step the DNA needs to enter the nucleus. It has been shown that the nuclear envelope represents a serious obstacle for the entry of pDNA into the nucleus, especially in non-dividing cells [4,5]. Therefore, in an attempt to bypass the nuclear envelope we investigated the possibility of transfecting cells with mRNA instead of pDNA. Besides the fact that nuclear delivery is not required for mRNA to be effective, mRNA-mediated transfection may hold several other advantages as well. Most importantly, there is no danger of introducing irreversible genomic modification. Moreover, in contrast to plasmid DNA, messenger RNA is devoid of immunogenic CpG motifs. Since the first study reported by Malone and colleagues [6] surprisingly little attention has been paid to the use of messenger RNA (mRNA) for the transfection of cells, which may be explained by the wide-held belief that mRNA is too unstable to ever be used as a drug. So far, only few studies focused on complexation of mRNA with cationic carriers (like cationic lipids and cationic polymers) in the last decade. As a consequence knowledge concerning mRNA-mediated transfection is still very scarce [7–12]. In addition to the use of cationic lipids and polymers also electroporation turned out to be a useful method for mRNA delivery to human embryonic and hematopoietic cells in vitro [13–15]. Successful mRNA-based applications will require sufficient knowl- edge on kinetics of the production of the protein of interest. The desired time frame of protein production will depend on the cell type to be transfected and the specific application. It is to be expected that both the onset and duration of protein expression will strongly depend on the nature of the mRNA carrier used as well as on the way the carrier/mRNA complexes are prepared. It is precisely for this reason that we set out to perform the research described in this paper: characterization of the transfection potential of mRNA complexes formed from mRNA and cationic lipids and polymers (Lipofectamine, DOTAP/DOPE, linear PEI), including a direct comparison with pDNA. 2. Materials and methods 2.1. Cell culture HeLa cells were cultured in Dulbecco's Modified Eagle's culture medium containing the growth factor F12 and phenol red (DMEM: F12) and supplemented with 2 mM glutamine, 10% heat-inactivated FBS and 100 U/ml penicillin/streptomycin. mMSC (a kind gift from Journal of Controlled Release 147 (2010) 385–391 ⁎ Corresponding author. Tel.: +32 9 264 80 78; fax: +32 9 264 81 89. E-mail address: stefaan.desmedt@ugent.be (S.C. De Smedt). 1 These authors equally contributed to the work. 0168-3659/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2010.07.124 Contents lists available at ScienceDirect Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel GENE DELIVERY