Design of peptide-targeted liposomes containing nucleic acids |
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Authors: | Adriana O. Santos,Lí gia C. Gomes da Silva,Luí s M. Bimbo,Maria C. Pedroso de Lima,Sé rgio Simõ es,Joã o N. Moreira |
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Affiliation: | a Laboratory of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra, Portugal b Center for Neuroscience and Cell Biology, University of Coimbra, Portugal c Department of Biochemistry, Faculty of Sciences and Technology, University of Coimbra, Portugal |
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Abstract: | Anticancer systemic gene silencing therapy has been so far limited by the inexistence of adequate carrier systems that ultimately provide an efficient intracellular delivery into target tumor cells. In this respect, one promising strategy involves the covalent attachment of internalizing-targeting ligands at the extremity of PEG chains grafted onto liposomes. Therefore, the present work aims at designing targeted liposomes containing nucleic acids, with small size, high encapsulation efficiency and able to be actively internalized by SCLC cells, using a hexapeptide (antagonist G) as a targeting ligand. For this purpose, the effect of the liposomal preparation method, loading material (ODN versus siRNA) and peptide-coupling procedure (direct coupling versus post-insertion) on each of the above-mentioned parameters was assessed. Post-insertion of DSPE-PEG-antagonist G conjugates into preformed liposomes herein named as stabilized lipid particles, resulted in targeted vesicles with a mean size of about 130 nm, encapsulation efficiency close to 100%, and a loading capacity of approximately 5 nmol siRNA/μmol of total lipid. In addition, the developed targeted vesicles showed increased internalization in SCLC cells, as well as in other tumor cells and HMEC-1 microvascular endothelial cells. The improved cellular association, however, did not correlate with enhanced downregulation of the target protein (Bcl-2) in SCLC cells. These results indicate that additional improvements need to be performed in the future, namely by ameliorating the access of the nucleic acids to the cytoplasm of the tumor cells following receptor-mediated endocytosis. |
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Keywords: | ODN, oligodeoxynucleotide SCLC, small cell lung cancer siRNA, small interfering RNA PEG, poly(ethylene glycol) CCL, coated cationic liposomes SALP, stabilized antisense lipid particles SNALP, stabilized nucleic acid lipid particles EGF, epidermal growth factor PFA, paraformaldehyde NaN3, sodium azide AD, actinomycin D 7-AAD, 7-aminoactinomycin D BSA, bovine serum albumin C12E8, octaethylene glycol monododecyl ether HSPC, hydrogenated soy phosphatidylcholine DSPC, 1,2-distearoyl-sn-glycero-3-phosphocholine CerC16-PEG, N-palmitoyl-sphingosine-1-succinyl(methoxypolyethylene glycol)2000 DSPE-PEG, N-palmitoyl-sphingosine-1-[succinyl(polyethylene glycol)]2000 DSPE-PEG-Mal, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)]2000 rhodamine-PE, L-α-phosphatidylethanolamine-N-(lissamine rhodamine B sulfonyl) DOTAP, 1,2-dioleyl-3-trimethylammonium-propane DODAP, 1,2-dioleyl-3-dimethylammonium-propane MES, 2-(n-morpholino)ethanesulfonic acid Tris, 2-amino-2-(hydroxymethyl)propane-1,3-diol HEPES, 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid CHOL, cholesterol MBS, MES buffered saline HBS, HEPES buffered saline SLP, stabilized lipid particles SLPODN, SLP encapsulating ODN SLPsiRNA, SLP encapsulating siRNA |
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