title: mRNA-Lipoplex loaded microbubble contrast agents for ultrasound-assisted transfection of dendritic cells.
abstract: In cancer immunotherapy the immune system should be triggered to specifically recognize and eliminate tumor cells in the patient's body. This could be achieved by loading dendritic cells (DCs) with tumor-associated antigens (TAAs). This can be achieved by transfecting DCs with messenger RNA encoding a tumor-associated antigen. Here we demonstrate transient transfection of dendritic cells by means of mRNA-lipoplexes bound to microbubbles. Microbubble-attached lipoplexes were introduced into the cells by applying ultrasound. Our data demonstrate that ultrasound-mediated delivery of mRNA-complexes led to efficient transfection of DCs. When mRNA encoding luciferase was used, maximal levels of the enzyme activity were detected 8 h after ultrasound application. Upon longer incubation protein expression gradually declined. This treatment did not affect viability of the cells. Intracellular localisation of mRNA-lipoplexes in DCs was determined by flow cytometry using fluorescently labeled lipoplexes. Over 50% of DCs contained fluorescently labeled mRNA-complexes. In the absence of additional maturation signals, transfection of immature DCs with mRNA-lipoplex loaded microbubbles and ultrasound application induced only a minor shift in the expression level of maturation markers (CD40 and CD86). However, in the presence of the activation stimulus (LPS), cells were able to further mature as shown by a significant up-regulation of CD40 expression. Thus, our results demonstrate that mRNA-lipoplex loaded microbubbles can serve as an applicable and safe tool for efficient mRNA-transfection of cultured DCs.
authors.affiliation: Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium.
authors.forename: Marie-Luce
authors.initials: ML
authors.lastname: De Temmerman
authors.forename: Heleen
authors.initials: H
authors.lastname: Dewitte
authors.forename: Roosmarijn E
authors.initials: RE
authors.lastname: Vandenbroucke
authors.forename: Bart
authors.initials: B
authors.lastname: Lucas
authors.forename: Claude
authors.initials: C
authors.lastname: Libert
authors.forename: Jo
authors.initials: J
authors.lastname: Demeester
authors.forename: Stefaan C
authors.initials: SC
authors.lastname: De Smedt
authors.forename: Ine
authors.initials: I
authors.lastname: Lentacker
authors.forename: Joanna
authors.initials: J
authors.lastname: Rejman
mesh_terms: D051940:B7-2 Antigen / Q000235:genetics; D019013:CD40 Antigens / Q000235:genetics; D002478:Cells, Cultured; D003287:Contrast Media / Q000737:chemistry*; D003713:Dendritic Cells / Q000378:metabolism*; D006801:Humans; D008055:Lipids / Q000737:chemistry; D045423:Microbubbles; D012333:RNA, Messenger / Q000008:administration & dosage* / Q000235:genetics; D014162:Transfection; D015854:Up-Regulation
chemical_list: D051940:B7-2 Antigen; D019013:CD40 Antigens; D003287:Contrast Media; D008055:Lipids; D012333:RNA, Messenger