Mineralized polyplexes for gene delivery: Improvement of transfection efficiency as a consequence of calcium incubation and not mineralization

Abstract

Gene therapy is an emerging field in which nucleic acids are used to control protein expression. The necessity of delivering nucleic acids to specific cell types and intracellular sites demands the use of highly specialized gene carriers. As a carrier modification technique, mineralization has been successfully used to modify viral and non-viral carriers, providing new properties that ultimately aim to increase the transfection efficiency. However, for the specific case of polyplexes used in gene therapy, recent literature shows that interaction with calcium, a fundamental step of mineralization, might be effective to increase transfection efficiency, leaving an ambiguity about of the role of mineralization for this type of gene carriers. To answer this question and to reveal the properties responsible for increasing transfection efficiency, we mineralized poly(aspartic acid) coated polyplexes at various CaCl2 and Na3PO4 concentrations, and evaluated the resultant carriers for physicochemical and morphological characteristics, as well as transfection and delivery efficiency with MC3T3-E1 mouse osteoblastic cells. We found that both mineralization and calcium incubation positively affected the transfection efficiency and uptake of polyplexes in MC3T3-E1 cells. However, this effect originated from the properties achieved by polyplexes after the calcium incubation step that are maintained after mineralization, including particle size increase, improved pDNA binding, and adjustment of zeta potential. Considering that mineralization can be a longer process than calcium incubation, we find that calcium incubation might be sufficient and preferred if improved transfection efficiency in vitro is the only effect desired.