A matrix reservoir for improved control of non-viral gene delivery.
ABSTRACT Non-viral gene delivery suffers from a number of limitations including short transgene expression times and low transfection efficiency. Collagen scaffolds have previously been investigated as in vitro DNA reservoirs, which allow sustained release of genetic information. Efficient viral gene-transfer from these scaffolds has previously been demonstrated. However, due to concerns about the safety of viral gene therapy, the use of non-viral vectors may be preferable. In this study a DNA-dendrimer complex embedded in a cross-linked collagen scaffold was investigated as a reservoir for non-viral delivery. Elution from the scaffolds and transfection of seeded rat mesenchymal stem cells were used to evaluate the scaffold's ability to act as a reservoir for the complexes. Elution from the scaffolds was minimal after 2 days with a total of 25% of the complexes released after 7 days. Extended transgene expression after DNA-dendrimer complex delivery from the scaffolds in comparison to direct delivery to cells was observed. The elongated transfection period and relatively high levels of reporter gene expression are significant advantages over other non-viral gene therapy techniques. This platform has the potential to be an effective method of scaffold-mediated gene delivery suitable for in vitro and in vivo applications.
- [Show abstract] [Hide abstract]
ABSTRACT: A poly (lactide-co-glycolide) (PLGA) scaffold filled with fibrin gel, mesenchymal stem cells (MSCs) and poly(ethylene oxide)-b-poly (L-lysine) (PEO-b-PLL)/pDNA-TGF-β1 complexes was fabricated and applied in vivo for synchronized regeneration of cartilage and subchondral bone. The PEO-b-PLL/pDNA-TGF-β1 complexes could transfect MSCs in vitro to produce TGF-β1 in situ and up regulate the expression of chondrogenesis-related genes in the construct. The expression of heterogeneous TGF-β1 in vivo declined along with the prolongation of implantation time, and lasted for 3 and 6 weeks in the mRNA and protein levels, respectively. The constructs (Experimental group) of PLGA/fibrin gel/MSCs/(PEO-b-PLL/pDNA-TGF-β1 complexes) were implanted into the osteochondral defects of rabbits to restore the functional cartilages, with gene-absent constructs as the Control. After 12 weeks, the Experimental group regenerated the neo-cartilage and subchondral bone with abundant deposition of glycosaminoglycans (GAGs) and type II collagen. The regenerated tissues had good integration with the host tissues too. By contrast, the defects were only partially repaired by the Control constructs. qRT-PCR results demonstrated that expression of the chondrogenesis-marker genes in the Experimental group was significantly higher than that of the Control group, and was very close to that of the normal cartilage tissue. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.Journal of Biomedical Materials Research Part A 03/2013; · 2.83 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: This study centers on the use of a nanoparticle based on the polysaccharide from Angelica sinensis (ASP) as an efficient and safe non-viral gene vector. After modification with branched low molecular weight polyethylenimine (1200 Da), the cationized ASP (cASP) was combined with the plasmid encoding transforming growth factor-beta 1 (TGF-β1) to form a spherical nano-scaled particle (i.e., cASP-pTGF-β1 nanoparticle). This nanoparticle was applied to transfect rat bone marrow mesenchymal stem cells and human umbilical cord mesenchymal stem cells. As a result, nanoparticles (cASP/pDNA weight ratio 10:1) had the greatest transfection efficiency in both cells, which was significantly higher than those of Lipofectamine2000 and PEI (25 kDa). This was in agreement with the findings of the semi-quantitative RT-PCR and live cell imaging. These nanoparticles were also less toxic than Lipofectamine2000 and PEI (25 kDa). Therefore, cASP could be a potential candidate for a novel non-viral gene vector.Nanomedicine: nanotechnology, biology, and medicine 05/2013; · 6.93 Impact Factor
- 04/2013: pages 315-357; , ISBN: ISBN 978-94-007-6009-7