Oligo-guanidyl targeted bioconjugates forming rod shaped polyplexes as a new nanoplatform for oligonucleotide delivery

J Control Release. 2019 Sep 28;310:58-73. doi: 10.1016/j.jconrel.2019.08.005. Epub 2019 Aug 7.


Novel bioconjugates (Agm6-M-PEG-FA) for active oligonucleotide (ON) delivery have been developed by conjugating a cationic oligo-guanidyl star-like shaped "head" (Agm6-M) to a polymeric "tail" (PEG) terminating with folic acid (FA) as targeting agent or methoxy group (Agm6-M-PEG-FA and Agm6-M-PEG-OCH3, respectively). Gel electrophoresis showed that the bioconjugates completely associated with ONs at 3 nitrogen/phosphate (N/P) ratio. Studies performed with folate receptor (FR)-overexpressing HeLa cells, showed that optimal cell up-take was obtained with the 75:25 w/w Agm6-M-PEG-OCH3:Agm6-M-PEG-FA mixture. Dynamic light scattering and transmission electron microscopy showed that the polyplexes had size <80 nm with narrow polydispersity and rod-shaped morphology. The polyplexes were stable for several hours in plasma while ON was released in the presence of heparin concentration 16-times higher than the physiological one. The polyplexes displayed negligible cytotoxicity, hemolysis and low pro-inflammatory TNF-α release. Studies performed with FR-overexpressing HeLa and MDA-MB-231 cells using siRac1 revealed that the folated polyplexes caused significantly higher gene silencing (86.1 ± 9.6%) and inhibition of cell migration (40%) than the non-folated polyplexes obtained with Agm6-M-PEG-OCH3 only. Although cytofluorimetric analyses showed similar cell uptake for both folated and non-folated polyplexes, confocal, TEM and competition studies showed that the folated polyplexes were taken-up by lysosome escaping caveolin-mediated pathway with final polyplex localization within cytosol, while non-folated polyplexes were preferentially taken-up via clathrin-mediated pathway to localize in the lysosomes. Finally, preliminary in vivo studies carried out in mice revealed that the folated polyplexes dispose in the tumor mass.

Keywords: Active targeting; Endocytotic pathways; Folate receptor; Oligonucleotide delivery; Polycationic polymers; Polyplexes; Supramolecular carriers; siRNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding, Competitive
  • Cell Line, Tumor
  • Cell Movement / drug effects
  • Cell Movement / genetics
  • Cell Survival / drug effects
  • Cell Survival / genetics
  • Folic Acid Transporters / genetics
  • Folic Acid Transporters / metabolism*
  • Gene Silencing
  • Gene Transfer Techniques*
  • HeLa Cells
  • Humans
  • Mice, Nude
  • Nanoconjugates / chemistry*
  • Neoplasms, Experimental / genetics
  • Neoplasms, Experimental / therapy
  • Oligonucleotides / administration & dosage*
  • Oligonucleotides / genetics
  • Particle Size
  • Protein Binding
  • Surface Properties
  • Xenograft Model Antitumor Assays


  • Folic Acid Transporters
  • Nanoconjugates
  • Oligonucleotides