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- Gene delivery using dendrimer/pDNA complexes immobilized in electrospun fibers using the Layer-by-Layer techniquePublication . Ramalingam, Kirthiga; Castro, Rita; Pires, Pedro; Shi, Xiangyang; Rodrigues, João; Xiao, Shili; Tomás, HelenaA gene delivery platform for potential use in tissue engineering applications was developed by surface functionalization of biodegradable electrospun poly(lactic-co-glycolic acid) (PLGA) fibers with nanolayers of chitosan (cationic polymer) and alginate (anionic polymer) using the Layer-by-Layer (LbL) technique. The developed system not only supported the attachment and growth of human Mesenchymal Stem Cells (hMSCs), but also was capable of delivering pDNA/dendrimer complexes and inducing cell differentiation towards the osteogenic lineage when a pDNA codifying for human Bone Morphogenetic Protein-2 (BMP-2) was used. Beyond providing a means for pDNA/dendrimer complex immobilization, the polyelectrolyte coating conferred sustained release properties to the scaffold that resulted in pDNA protection from degradation. The polyelectrolyte coating, by itself, also contributed to enhance cell differentiation.
- Fine tuning of the pH-sensitivity of laponite–doxorubicin nanohybrids by polyelectrolyte multilayer coatingPublication . Xiao, Shili; Castro, Rita; Maciel, Dina; Gonçalves, Mara; Shi, Xiangyang; Rodrigues, João; Tomás, HelenaDespite the wide research done in the field, the development of advanced drug delivery systems with improved drug delivery properties and effective anticancer capability still remains a great challenge. Based on previous work that showed the potentialities of the nanoclay Laponite as a pH-sensitive doxorubicin (Dox) delivery vehicle, herein we report a simple method to modulate its extent of drug release at different pH values. This was achieved by alternate deposition of cationic poly(allylamine) hydrochloride and anionic poly(sodium styrene sulfonate) (PAH/PSS) polyelectrolytes over the surface of Dox-loaded Laponite nanoparticles using the electrostatic layer-by-layer (LbL) self-assembly approach. The successful formation of polyelectrolyte multilayer-coated Dox/Laponite systems was confirmed by Dynamic Light Scattering and zeta potential measurements. Systematic studies were performed to evaluate their drug release profiles and anticancer efficiency. Our results showed that the presence of the polyelectrolyte multilayers improved the sustained release properties of Laponite and allowed a fine tuning of the extension of drug release at neutral and acidic pH values. The cytotoxicity presented by polyelectrolyte multilayer-coated Dox/Laponite systems towards MCF-7 cells was in accordance with the drug delivery profiles. Furthermore, cellular uptake studies revealed that polyelectrolyte multilayer-coated Dox/Laponite nanoparticles can be effectively internalized by cells conducting to Dox accumulation in cell nucleus.
- PAMAM dendrimer/pDNA functionalized-magnetic iron oxide nanoparticles for gene deliveryPublication . Xiao, Shili; Castro, Rita; Rodrigues, João; Shi, Xiangyang; Tomás, HelenaHerein, we report an easy and ingenious method to functionalize magnetic iron oxide nanoparticles (MNPs) with plasmid DNA (pDNA) to obtain nanohybrid systems suitable for nucleic acid therapy. The nanohybrids were prepared by combining complexes of dendrimers and pDNA (dendriplexes) and poly(styrene) sulfonate-coated MNPs through electrostatic interactions. The effects of the dendrimer generation (generations 2, 4 and 6) and the amine to phosphate group (N/P) ratio on the hydrodynamic diameter, zeta potential, cell viability, cellular internalization and transfection efficiency of the nanohybridswere systematically investigated at different transfection conditions (including incubation time, pDNA concentration, presence or absence of an external magnetic field, and presence or absence of fetal bovine serum). The results confirmed that the nanohybrids were able to transfect NIH 3T3 cells, and that the level of gene expression (the luciferase protein reporter gene was used) was strongly dependent on the dendrimer generation, the N/P ratio, and the pDNA concentration. The best system was based on dendriplex-coated MNPs formed by generation 6 dendrimers at an N/P ratio of 10 that, at optimized conditions, led to a gene expression level which was not significantly different from that obtained only using dendriplexes. In summary, a coherent set of results was reached indicating the potential of the developed nanohybrids as effective gene delivery nanomaterials.
- Electrospun laponite-doped poly(lactic-co-glycolic acid) nanofibers for osteogenic differentiation of human mesenchymal stem cellsPublication . Wang, Shige; Castro, Rita; An, Xiao; Song, Chenlei; Luo, Yu; Shen, Mingwu; Tomás, Helena; Zhu, Meifang; Shi, XiangyangWe report the fabrication of uniform electrospun poly(lactic-co-glycolic acid) (PLGA) nanofibers incorporated with laponite (LAP) nanodisks, a synthetic clay material for osteogenic differentiation of human mesenchymal stem cells (hMSCs). In this study, a solution mixture of LAP suspension and PLGA was electrospun to form composite PLGA–LAP nanofibers with different LAP doping levels. The PLGA–LAP composite nanofibers formed were systematically characterized via different techniques. We show that the incorporation of LAP nanodisks does not significantly change the uniform PLGA fiber morphology, instead significantly improves the mechanical durability of the nanofibers. Compared to LAP-free PLGA nanofibers, the surface hydrophilicity and protein adsorption capacity of the composite nanofibers slightly increase after doping with LAP, while the hemocompatibility of the fibers does not appreciably change. The cytocompatibility of the PLGA–LAP composite nanofibers was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay of L929 mouse fibroblasts and porcine iliac artery endothelial cells cultured onto the surface of the nanofibers. The results reveal that the incorporated LAP is beneficial to promote the cell adhesion and proliferation to some extent likely due to the improved surface hydrophilicity and protein adsorption capability of the fibers. Finally, the PLGA–LAP composite nanofibers were used as scaffolds for osteogenic differentiation of hMSCs. We show that both PLGA and PLGA–LAP composite nanofibers are able to support the osteoblast differentiation of hMSCs in osteogenic medium. Most strikingly, the doped LAP within the PLGA nanofibers is able to induce the osteoblast differentiation of hMSCs in growth medium without any inducing factors. The fabricated smooth and uniform organic–inorganic hybrid LAP-doped PLGA nanofibers may find many applications in the field of tissue engineering.