Abstract
AbstractSafe and efficient gene delivery vectors will enhance the prospects for polynucleotide‐based therapies. Herein a new approach toward structurally optimized gene vector design based on the preparation of clickable poly(allylamino‐phosphazene)s that can be converted to several cationic and anionic derivatives via thiol–ene addition is described. Simultaneous co‐incubation of alkylamine‐ and alkylcarboxylate‐poly(phosphazenes) with polynucleotide generates binary polyelectrolyte nanoparticles. Screening of a series of these complexes for transfection in glioblastoma cells shows that the inclusion of 6‐mercaptohexanoic acid substituted poly(phosphazene)s in the complexes results in six‐fold and 19‐fold higher luciferase expression in U87MG cells and GBM1 primary cells, respectively. This effect is attributed to the specific ionization properties of these materials that improved polyplex intracellular trafficking. Transfection in 3D‐spheroid models and subcutaneous xenograft U87MG tumors confirms higher transgene expression for the binary cationic/anionic poly(phosphazene) complexes compared to the related polycation‐pDNA complexes and to PEI‐pDNA complexes. The data also indicate a notable capacity of the mixed complexes to deliver genes to the inner cores of tumor spheroids. Extension of this approach to siRNA delivery shows that the mixed poly(phosphazene) complexes can silence DYRK1A, a gene implicated in glioblastoma initiation and progression, reducing U87MG cell renewal in vitro and delaying tumor growth in vivo.
Highlights
Therapies based on polynucleotides offer much promise but require more efficient and safer gene carriers [1]
When preparing polymers for gene delivery, this synthetic route is problematic because most functional candidates display more than two nucleophilic centers, which can result in general crosslinking of the material during derivatization reactions leading to precipitation of the partially substituted polymer
The initially formed poly(dichlorophosphazene) (2) was characterized by 31P-Nuclear Magnetic Resonance Spectroscopy (NMR) (Figure S1), and this compound was reacted by nucleophilic substitution at the P-Cl bonds with allylamine to generate the key precursor polymer, allylamino-polyphosphazene (AAPPZ)
Summary
Therapies based on polynucleotides offer much promise but require more efficient and safer gene carriers [1]. We have developed a poly(phosphazene) platform, containing side-chain double-bond repeating units, and prepared a series of cationic and anionic derivatives of these via thiol-ene grafting chemistries using -aminoalkanethiols and carboxylatoalkanethiols. By combining these materials in the presence of nucleic acids, we aimed to generate mixed polyelectrolyte complexes with sufficient positive charge to bind polynucleotides and promote cell internalization, but with the capacity to destabilize cell membranes in response to pH, and at the same time to minimize polycation-induced toxicity. These optimized complexes were expected to escape more effectively than conventional polycation/nucleic acid complexes from the endolysosomal compartment, which has been considered to be the most significant intracellular barrier to effective gene delivery [3b, 7]
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