The healing potential of scaffolds for tissue engineering can be enhanced by combining them with genes to produce gene-activated matrices (GAMs) for tissue regeneration. We examined the potential of using polyethyleneimine (PEI) as a vector for transfection of mesenchymal stem cells (MSCs) in monolayer culture and in 3D collagen-based GAMs. PEI-pDNA polyplexes were fabricated at a range of N/P ratios and their optimal transfection parameters (N/P 7 ratio, 2μg dose) and transfection efficiencies (30±8%) determined in monolayer culture. The polyplexes were then loaded onto collagen, collagen-glycosaminoglycan and collagen-nanohydroxyapatite scaffolds where gene expression was observed up to 21 days with a polyplex dose as low as 2μg. Transient expression profiles indicated that the GAMs act as a polyplex depot system whereby infiltrating cells become transfected over time as they migrate throughout the scaffold. The collagen-nHa GAM exhibited the most prolonged and elevated levels of transgene expression. This research has thus demonstrated that PEI is a highly efficient pDNA transfection agent for both MSC monolayer cultures and in the 3D GAM environment. By combining therapeutic gene therapy with highly engineered scaffolds, it is proposed that these GAMs might have immense capability to promote tissue regeneration.

A series of well-defined star-shaped polypeptides were successfully synthesised by the ring opening polymerisation (ROP) of the N-carboxyanhydride (NCA) of ε-carbobenzyloxy-L-lysine (ZLL) using a range of generations of polypropylene imine (PPI) dendrimers as multifunctional initiators. The monomer feed ratio and dendrimer generation were varied to afford a series of polypeptide dendrimer hybrids with superior structural versatility and functionality. Subsequent protecting group removal yielded star-shaped poly(lysine) of controlled variation in polypeptide chain length and arm multiplicity. Star-shaped PLL polymers were used to prepare pDNA and siRNA to form “polyplexes” to determine their ability to complex different nucleic acid cargoes and were compared with linear PLL polyplex controls. Significant differences in size and surface charge were seen between star-shaped PLL polyplexes and linear PLL polyplexes for both cargoes. The star-shaped polypeptides were capable of more effective complexation of both nucleic acids at low N/P ratios compared to linear PLL as evidenced by zeta potential and electrophoretic data. This was particularly evident in siRNA polyplexes as linear PLL failed to completely complex siRNA into nanocomplexes of appropriate size for cell transfection i.e. <200 nm in size, while star poly(lysine) formed siRNA polyplexes <100 nm at certain N/P ratios, albeit strongly dependent on the particular molecular weight and architecture, as analysed by dynamic light scattering (DLS). Atomic force microscopy (AFM) identified discrete spherically shaped polyplexes for all star-shaped polypeptide-based polyplexes while linear PLL formed elongated irregular shaped complexes. This difference in morphology may go some way towards explaining the 300-fold increase in luciferase expression seen for star-shaped PLL polyplexes G5(64)-PLL₄₀ compared to linear PLL pGLuc polyplexes in epithelial cells. Each of the PPI-PLL polymers appeared to be capable of protecting the nucleic acid cargoes from degradation by the relevant nuclease enzyme as effectively as the positive control polyethyleneimine (PEI) polyplexes. Overall the promising nucleic acid complexation, sizing, morphology and protection capacity of two different genetic “cargoes” highlight the potential of polypeptide dendrimer hybrids as gene delivery vectors.