Delivery of non viral gene carriers from sphere templated fibrin scaffolds for sustained transgene expression

Background:

The scaffolds being developed are for replacement of soft tissues. The ideal scaffold is one that has similar mechanical properties as the replaced tissue, and allows for biochemical cues to be integrated to directed the cellular processes and create the necessary organization. Many other scaffolds have controlled rates of degradation which allows for time dependent delivery of growth factors, however special preparation is needed to prevent premature degradation and denaturation. There are some scaffold materials, such as chitosan, collagen, fibrin, or composites that can couple the growth factors to the scaffold via side chain reactions, but these materials generally lack the mechanical strength required for soft tissue replacement, and are broken down too quickly in vivo. Thus, Pun et al decided to deliver the genes necessary for the production of the needed growth factors, instead of the growth factors themselves. Because DNA retains its structural and functional integrity better than proteins and growth factors, pun’s method makes this method compatible with more scaffold preparation methods.

The polymer used, (PEI), forms complexes with DNA to form nanoparticles on the range of 50-200nm in diameter. They are non-immunogenic and are taken up by cells through endocytosis, mediated by electrostatic association between positively charged polyplexes and negatively charged cell surfaces. When the nanoparticle enters the cell, it is hypothesized that it disrupts the endosome by the “proton sponge effect”, which is basically the ability to buffer the endosome and cause its rupture. The DNA will then, by unknown methods, make its way to the nucleus where it then engages in transcription and translation of the desired gene products. The risks associated with viral vectors, such as recombination and mutagenesis are avoided with this nanoparticle approach.

Summary of results:

Pun et al used a three-dimensional, sphere-templated fibrin scaffold to deliver controlled levels of gene delivery vectors. The scaffold was developed by Pun et al such that it has mechanical properties similar to soft tissues. The gene delivery vectors are polymeric nanoparticles, known as polylexes (In this paper, they used polyethylenimine (PEI)). The polyplexes were embedded within the scaffolds using two different methods and the resulting gene delivery and expression profiles are given. One method is embedding the polyplexes within the scaffold, the other method is only coating the polyplexes on the surface. The embedding technique allows for slower sustained delivery, whereas the surface coated approach allowed for much more rapid uptake.

They noticed aggregation of the polyplexes and attempted to pegylate the surfaces of the polyplexes to reduce aggregation. Although it was successful to some extent, this reduced the transfection rate.

The scaffolds tested were prepared by the sphere-templating technique and were fabricated with the dimensions of 2mm thickness by 8mm diameter. The scaffolds were imaged and given an estimated porosity of 74.7 +/- 0.6%, determined by digital volumetric imaging. They mention that further optimization of the porosity is needed.

The expression of the gene delivered VEGF, cell viability relative to a fibrin scaffold control, DNA release from the scaffolds, and long term expression of the delivered hVEGF plasmid were all measured and results are described in the following figures. The cell type used in the experiment were mouse 3T3 fibroblasts.

Figure 1: (A) is a schematic illustration of the sphere template fibrin scaffold production procedure with surface coated or embedded polyplexed or uncomplexed DNA. (B) This is what the distribution of the polyplexes is hypothesized to be like with relation to the fibrin scaffold. The schematic is of a single pore of the sphere template. The blue spheres are the polyplexes and the red spheres are the fibrillar matrix of fibrin.

Figure 2: These graphs demonstrate the release kinetics of the DNA from the scaffold. The diamonds are the surface coated DNA, the surface coated polyplexes of carious N:P ratios are: (circles = 5, triangles = 10, squares = 20). The unfilled diamonds are the embedded DNA, and the unfilled squares are the embedded polyplexes. These graphs demonstrate that the DNA dissociates rapidly from the scaffold, and that the embedded polyplexes adhere strongly to the scaffold.

Figure 3: These graphs depict the long term transgene expression of the hVEGF plasmid, determined by an ELISA assay. (A) Shows the surface coated polyplexes of N:P = 20. These have a peak expression level at day 5, and detectable expression through day 27. (B) Shows cumulative VEGF expression of the same scaffolds in (A). It shows a linear expression through 15 days. (C) Shows the polyplexes embedded with scaffolds. They show peak transfection at day 9. (N:P = 10, gray bars) or at day 7 (N:P = 20, black bars). (D) Shows the cumulative VEGF expression, which shows linear expression through day 29. (N:P = 10, unfilled squares) or through day 21 (N:P = 20, filled squares). The scaffolds with no DNA showed no detectable VEGF expression. (The filled diamonds).

Figure 4: This graph shows the long term cell viability with time on the fibrin scaffolds. The data was normalized to the viability of cells on plain fibrin scaffolds. (filled diamonds) The polyplexes embedded within the scaffolds are (N:P = 20, filled squares). They didn’t show a significant decrease in cell viability. The surface coated polyplexes (N:P = 20, unfilled squares) had a significantly lower viability at days 3, 7, and 14. At day 27, the viability was similar for all of the scaffolds.

Why this paper is pretty awesome:

This non-viral gene delivery method is a nice alternative to the traditional scaffold + growth factor/protein approach. The gene delivery method itself is very nice in that it is non-immunogenic and doesn’t cause recombination and insertion into the cell’s genome like many viral vectors do. Although polymeric nanoparticle gene delivery methods are usually not as effective as other methods, this paper demonstrates that it is sufficient for tissue engineering applications, but I'd like to see it be better.