Gene therapy in combination with an approach targeting the tumour microenvironment in glioblastoma

The objective of this work is to develop glycol chitosan (GC) based gene delivery vehicles with an extracellular matrix (ECM) targeting capacity incorporating hyaluronidase as part of a three-component electrostatic complex of a polymer, a plasmid and an enzyme to improve the diffusion of the nanosystems in the tumor microenvironment. This is also to the best of our knowledge the first study, describing a quantitative strategy using an HPLC method to estimate the amount of bound enzyme to the ternary electrostatic complex. Our data showed that molecular weight, polymer to DNA mass ratio, treatment and complexation time as well as complexation medium all influence the potential of the nanosystems to deliver genes in the U87 glioma cell line in vitro. When compared to the positive control, lipofectamine a GC polymer of 37 kDa (GC37) showed about 70 % of lipofectamine’s transfection efficiency to deliver a β-Gal reporter gene to U87 cells after 17 h treatment time and 24 h complexation time at 4° C. The ternary electrostatic complexes were ineffective at delivering β-Gal DNA in vitro, although they showed some transfection potential in mice on nasal administration. However further studies are needed to confirm these findings and to identify the brain regions where the β-Gal protein is expressed. A subcutaneous and an intranasal route of administration were used to test the in vivo transfection efficiency of the GC37 polyplexes in mice and to compare it to the positive control lipofectamine and naked β-Gal. Lipofectamine/β-Gal lipoplexes (LF2) appeared with the highest signal measured by IVIS, 48 h post subcutaneous administration when compared to the naked plasmid (D2) and the GC37/β-Gal polyplex (GCP3) all at the same dose (0.207 μg kg -1 β-Gal DNA), although statistical analysis identified no significant differences between any of the treatments. On the other hand, the main intranasal study showed baseline levels of active β-Gal enzyme for mice treated with lipofectamine/ β-Gal lipoplexes (LF1), naked β-Gal DNA (D1) and GC37/β-Gal polyplexes (GCP2) all at the same dose (0.067 mg kg-1 β-Gal DNA). This raised important questions, which need addressing such as the suitability of the enzyme activity assay for nasal administration along with reproducibility concerns surrounding nasal administration in mice. Although further studies are needed to reveal the full in vivo potential of GC37 for effects occurring away from the administration site, GC37 could offer a considerable advantage as an attractive candidate for polymer-mediated gene therapy due to its significantly lower cytotoxicity than lipofectamine and comparable transfection potential.