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- Non-viral gene delivery to mesenchymal stem cells: methods, strategies and application in bone tissue engineering and regenerationPublication . Santos, José L.; Pandita, Deepti; Rodrigues, João; Pêgo, Ana P.; Granja, Pedro L.; Tomás, HelenaMesenchymal 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.
- Receptor-mediated gene delivery using PAMAM dendrimers conjugated with peptides recognized by mesenchymal stem cellsPublication . Santos, José L.; Pandita, Deepti; Rodrigues, João; Pêgo, Ana P.; Granja, Pedro L.; Balian, Gary; Tomás, HelenaAs 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.
- Functionalization of poly(amidoamine) dendrimers with hydrophobic chains for improved gene delivery in mesenchymal stem cellsPublication . Santos, José L.; Oliveira, Hugo; Pandita, Deepti; Rodrigues, João; Pêgo, Ana P.; Granja, Pedro L.; Tomás, HelenaA new family of gene delivery vectors is synthesized consisting of a medium-size generation PAMAM dendrimer (generation 5, with amine termini) core randomly linked at the periphery to hydrophobic chains that vary in length (12 to 16 carbon alkyl chains) and number (from 4.2 to 9.7 in average). The idea subjacent to the present work is to join the advantages of the cationic nature of the dendrimer with the capacity of lipids to interact with biological membranes. Unlike other amphiphilic systems designed for the same purpose, where the hydrophobic and hydrophilic moieties coexist in opposite sides, the present vectors have a hydrophilic interior and a hydrophobic corona. The vectors are characterized in respect to their ability to neutralize, bind and compact plasmid DNA (pDNA). The complexes formed between the vectors and pDNA are analyzed concerning their size, zeta-potential, resistance to serum nucleases, capacity of being internalized by cells and transfection efficiency. These new vectors show a remarkable capacity for mediating the internalization of pDNA with minimum cytotoxicity, being this effect positively correlated with the -CH(2)- content present in the hydrophobic corona. Gene expression in MSCs, a cell type with relevancy in the regenerative medicine clinical context, is also enhanced using the new vectors but, in this case, the higher efficiency is shown by the vectors containing the smallest hydrophobic chains.