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Gene delivery into mesenchymal stem cells: a biomimetic approach using RGD nanoclusters based on poly(amidoamine) dendrimers

Publication . Pandita, Deepti; Santos, José L.; Rodrigues, João; Pêgo, Ana P.; Granja, Pedro L.; Tomás, Helena

Poly(amidoamine) dendrimers (generations 5 and 6) with amine termini were conjugated with peptides containing the arginine-glycine-aspartic acid (RGD) sequence having in view their application as gene delivery vectors. The idea behind the work was to take advantage of the cationic nature of dendrimers and of the integrin targeting capabilities of the RGD motif to improve gene delivery. Dendrimers were used as scaffolds for RGD clustering and, by controlling the number of peptides (4, 8, and 16) linked to each dendrimer, it was possible to evaluate the effect of RGD density on the gene delivery process. The new vectors were characterized in respect to their ability to neutralize and compact plasmid DNA (pDNA). The complexes formed by the vectors and pDNA were studied concerning their size, zeta potential, capacity of being internalized by cells and ability of transferring genes. Transfection efficiency was analyzed, first, by using a pDNA encoding for Enhanced Green Fluorescent Protein and Firefly Luciferase and, second, by using a pDNA encoding for Bone Morphogenetic Protein-2. Gene expression in mesenchymal stem cells was enhanced using the new vectors in comparison to native dendrimers and was shown to be dependent on the electrostatic interaction established between the dendrimer moiety and the cell surface, as well as on the RGD density of nanoclusters. The use of dendrimer scaffolds for RGD cluster formation is a new approach that can be extended beyond gene delivery applications, whenever RGD clustering is important for modulating cellular responses.

2011-02-14Journal article

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Non-viral gene delivery to mesenchymal stem cells: methods, strategies and application in bone tissue engineering and regeneration

Publication . Santos, José L.; Pandita, Deepti; Rodrigues, João; Pêgo, Ana P.; Granja, Pedro L.; Tomás, Helena

Mesenchymal stem cells (MSCs) can be isolated from several tissues in the body, have the ability to selfrenewal, show immune suppressive properties and are multipotent, being able to generate various cell types. At present, due to their intrinsic characteristics, MSCs are considered very promising in the area of tissue engineering and regenerative medicine. In this context, genetic modification can be a powerful tool to control the behavior and fate of these cells and be used in the design of new cellular therapies. Viral systems are very effective in the introduction of exogenous genes inside MSCs. However, the risks associated with their use are leading to an increasing search for non-viral approaches to attain the same purpose, even if MSCs have been shown to be more difficult to transfect in this way. In the past few years, progress was made in the development of chemical and physical methods for non-viral gene delivery. Herein, an overview of the application of those methods specifically to MSCs is given and their use in tissue engineering and regenerative medicine therapeutic strategies highlighted using the example of bone tissue. Key issues and future directions in non-viral gene delivery to MSCs are also critically addressed.

2011Journal article

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Osteogenic differentiation of mesenchymal stem cells using PAMAM dendrimers as gene delivery vectors

Publication . Santos, José Luís; Oramas, Elena; Pêgo, Ana Paula; Granja, Pedro Lopes; Tomás, Helena

This paper reports the use of different generations of polyamidoamine (PAMAM) dendrimers for the in vitro transfection of mesenchymal stem cells (MSCs). A systematic study was carried out on the transfection efficiency achieved by the PAMAM dendrimers using a beta-galactosidase reporter gene system. Transfection results were shown to be dependent upon the generation of dendrimers, the amine to phosphate group ratio and the cell passage number. In all cases, the transfection efficiency was very low. Nevertheless, it was hypothesized that a low transfection level could be sufficient to promote the in vitro differentiation of MSCs towards the osteoblastic lineage. To address this possibility, dendrimers carrying the human bone morphogenetic protein-2 (hBMP-2) gene-containing plasmid were used. All quantitative (alkaline phosphatase activity, osteocalcin secretion and calcium deposition) and qualitative (von Kossa staining) osteogenic markers were significantly stronger in transfected cells when compared to non-transfected ones. This study not only clearly demonstrates that a low transfection level can be sufficient for inducing in vitro differentiation of MSCs to the osteoblast phenotype but also highlights the importance of focusing research on the development of gene delivery vectors in the concrete application.

2009Journal article

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Receptor-mediated gene delivery using PAMAM dendrimers conjugated with peptides recognized by mesenchymal stem cells

Publication . Santos, José L.; Pandita, Deepti; Rodrigues, João; Pêgo, Ana P.; Granja, Pedro L.; Balian, Gary; Tomás, Helena

As mesenchymalstemcells(MSCs)candifferentiateintomultiplecelltypes,thedeliveryof exogenous genes to this type of cell can be an important tool in tissue regeneration and engineering. HowevertransfectionofMSCsusingnonviralgenedeliveryvectorsisdifﬁcult,thedevelopmentofmore efﬁcientandsafeDNAvehiclesbeingnecessary.Moreover,speciﬁctransfectionofMSCsmayberequired to avoid unwanted side effects in other tissues. In this study, a novel family of gene delivery vectors based on poly(amidoamine) (PAMAM) dendrimers functionalized with peptides displaying high afﬁnity toward MSCs was prepared. The vectors were characterized with respect to their ability to neutralize, bindandcompactplasmidDNA(pDNA).ThecomplexesformedbetweenthevectorsandpDNAwere analyzedconcerningtheirsize, -potential,capacityofbeinginternalizedbycellsandtransfectionefﬁciency. Thesenewvectorsexhibitedlowcytotoxicity,receptor-mediatedgenedeliveryintoMSCsandtransfection efﬁciencies superior to those presented by native dendrimers and by partially degraded dendrimers.

2010-06-07Journal article

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