Article
An antibody-guided poly-propylene-imine (PPI)-based polyplex-system for siRNA-treatment of EGFRvIII-positive tumors
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Published: | June 9, 2017 |
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Objective: Therapeutics based on small interfering RNAs (siRNAs) offer great potential to treat so far incurable diseases such as glioblastoma multiforme (GBM). However, the broad application of siRNAs using various non-viral carrier systems is hampered by unspecific toxic or immunogenic side effects, poor pharmacokinetics due to unwanted delivery of siRNA-loaded nanoparticles into non-target cells, as well as inefficient internalization into target cells. In order to overcome these obstacles, we have developed a single chain antibody fragment (scFv)-guided polyplex system for targeted delivery, based on transfection-disabled maltose-modified poly-propylene-imine siRNA carrier molecules. To achieve selective siRNA delivery into EGFRvIII-positive tumor cells, a specific anti-EGFRvIII single chain antibody (scFv(MR1.1)) was utilized and conjugated to polyplexes through a novel coupling strategy and evaluated in vitro an in tumor xenografts.
Methods: Polyplex formation was investigated by Western Blot analysis and atomic force microcopy. Targeted delivery of scFv(MR1.1)-P-BAP guided polyplexes to EGFRvIII-positive cells in vitro were analyzed by flow cytometry and confocal laser scanning microscopy. Subsequently, delivery of these polyplexes to EGRFvIII-positive tumors was tested in an in vivo setting using nude mice.
Results: The production of a scFv fused with a biotinylation acceptor peptide (P-BAP) sequence derived from Propionibacterium shermanii transcarboxylase in biotin ligase-expressing HEK293T cells leaded to functional mono-biotinylated scFv-P-BAPs. Polyplex formation was achieved by a sequential conjugation of scFv-P-BAP to NeutrAvidin and mono-biotinylated mal19-biotin at defined stoichiometry. Compared to polyplexes conjugated to an unspecific control scFv-P-BAP, the generated tumor-specific polyplexes were able to bind to EGFRvIII-positive target cells and to exclusively deliver siRNA by selective receptor-mediated endocytosis. Atomic force microscopy revealed stable polyplexes, with a mean diameter of 150 nm that circumvents fast renal excretion and therefore provided a further precondition for the specific accumulation of tumor-specific polyplexes in subcutaneous tumors of nude mice.
Conclusion: In our study, we established a novel biotin-NeutrAvidin-conjugation system for coupling scFvs or other genetically engineered antibodies/protein ligands to glycodendrimers. Furthermore our results suggest that receptor-mediated uptake of siRNA containing polyplexes are a promising approach to improve siRNA therapy of cancer, and introduce a novel strategy for treatment of GBM.