Mixed anionic and cationic polyphosphazene complexes for effective gene delivery to glioblastoma in vitro and in vivo

Gene delivery vectors that are safe, efficient and affordable could significantly enhance the prospects for genetic-based therapies. Here we describe an approach to such vectors, using new variants of polyphosphazene materials and describe the synthesis of a series of degradable polyphosphazenes (PPZ) with both cationic and anionic side-chains, and report their use as mixed polyelectrolyte complexes for DNA and RNA delivery. First of all, a strategy for syntheses of various substituted PPZ was via a simple vinylic side-chain PPZ. This precursor takes advantages on generating various derivatisation via thiol-ene addition, yielding a series of alkylamine-polycations and alkylcarboxylate-polyanions. Then, we co-formulate the different polycations and polyanions to form nanocomplexes as gene vectors. Compared with polycation-only complexes (diameter 80 nm, +40 mV), the mixed complexes (Polycation/polyanion/pDNA) presented a more compact nanoscale structure and a reduction of -10 mV in surface charge. Optimization in 2D monolayer U87MG concluded that 6-mercaptohexanoic acid substituted PPZ (6MHA-PPZ) mixed in the polycation CA-PPZ complex resulted in the highest luciferase expression in the cells. These data are consistent with the higher buffering capability within early endosomal pH range of 6MHA-PPZ in comparison with the other anionic side-chain polymers. For proof-of-concepts for endosomal escapes, we investigated intracellular tracking by confocal imaging, showing that presence of 6MHA-PPZ in the complex results in lower co-localization with endo/lysosomes. This efficient endosomal escape is probably linked to the capacity of 6MHA-PPZ to rupture lipid membranes in the acid environment of the endosomes as we have confirmed by haemolysis tests. Herein, the complexes integrating 6MHA-PPZ presented negligible haemolytic effect at neutral pH, but a haemolytic effect comparable to poly-L-lysine (PLL) at the acidic pH. Besides traditional 2D monolayer models, 3D spheroids are better able to provide tissue-like architecture and gene expression profiles. To date very few studies have investigated nucleic acid delivery in tumour spheroids, despite of the fact that they are perfectly suited as preliminary studies before in vivo. As observed by tomographic imaging, CA-/6MHA-PPZ complexes provided higher transfection in U87MG spheroids as compared to CA-PPZ:pDNA and the reference PEI:pDNA complexes. This observation could be related to the physicochemical properties of CA-/6MHA-PPZ complexes, with their partially neutralized cationic charges at physiological pH, which might shield the system from indiscriminate binding to the extracellular matrix in tumour spheroids. The same optimized formulation to deliver a siRNA sequence with known activity against glioblastoma initiating cells (against DYRK1A, siDYR). The CA-/6MHA-PPZ:siDYR formulation was evaluated in U87MG xenograft mice, in a therapeutic scheme including co-treatment of a first-line chemo-drug temozolamide. The experiment found a significant delay in tumour progression in the arm receiving TMZ and CA-/6MHA-PPZ:siDYR vs. the arm receiving TMZ and CA-/6MHA-PPZ complexed with a scrambled siRNA sequence. Therefore, our results are consistent with an additional therapeutic benefit in combining the CA-/6MHA-PPZ:siDYR gene therapy with the standard pharmacological GBM treatment. Further studies will be needed to elucidate the possible clinical impact of this co-therapy on more advanced models and performing additional phenotypic analysis in the tumours. Overall, the data established a new versatile, biodegradable polymeric gene delivery based on polyphosphazenes with remarkable capacity for transfection, tumour penetration and in vivo performance.