TY - JOUR
T1 - Oligo-guanidyl targeted bioconjugates forming rod shaped polyplexes as a new nanoplatform for oligonucleotide delivery
AU - Malfanti, Alessio
AU - Scomparin, Anna
AU - Pozzi, Sabina
AU - Gibori, Hadas
AU - Krivitsky, Adva
AU - Blau, Rachel
AU - Satchi-Fainaro, Ronit
AU - Mastrotto, Francesca
AU - Caliceti, Paolo
AU - Salmaso, Stefano
N1 - Publisher Copyright:
© 2019 Elsevier B.V.
PY - 2019/9/28
Y1 - 2019/9/28
N2 - 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.
AB - 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.
KW - Active targeting
KW - Endocytotic pathways
KW - Folate receptor
KW - Oligonucleotide delivery
KW - Polycationic polymers
KW - Polyplexes
KW - Supramolecular carriers
KW - siRNA
UR - http://www.scopus.com/inward/record.url?scp=85070672462&partnerID=8YFLogxK
U2 - 10.1016/j.jconrel.2019.08.005
DO - 10.1016/j.jconrel.2019.08.005
M3 - Article
C2 - 31400381
AN - SCOPUS:85070672462
SN - 0168-3659
VL - 310
SP - 58
EP - 73
JO - Journal of Controlled Release
JF - Journal of Controlled Release
ER -