Receptor-targeted liposome-peptide nanocomplexes for siRNA delivery

Aristides D Tagalakis, Lin He, Luisa Saraiva, Kenth T Gustafsson, Stephen L Hart

Research output: Contribution to journalArticle

58 Citations (Scopus)

Abstract

RNA interference induced by double-stranded, small interfering RNA (siRNA) molecules has attracted great attention as a genetic therapeutic approach. Despite major advances in this field, new nanoparticle formulations are required for in vivo delivery of siRNA, particularly for tissue-specific delivery of siRNA reagents. We have developed and optimized LYR nanocomplex formulations for siRNA delivery that consist of a liposome (DOTMA/DOPE; L) and a targeting peptide (K₁₆GACYGLPHKFCG; Y) which self-assemble on mixing at optimal ratios with siRNA (R). Biophysical measurements indicated that LYR nanocomplexes were strongly cationic, mainly spherical particles of less than 100 nm. These formulations packaged and protected siRNA on incubation with RNAseA with >90% intact siRNA recovery. In addition, intact siRNA was recovered from LYRs upon heparin treatment. A critical synergy was observed between the lipid and peptide components for LYR particle stability and transfection efficiency. To evaluate targeting, transfections were compared with non-targeted formulations containing K₁₆ with no targeting ligand. Gene knockdown efficiencies with targeted formulations were more than two-fold better in all cell lines tested (p < 0.01). LYR formulations with liposomes containing DOTMA, which has an 18-carbon (C18) alkyl tail, were significantly better in silencing than formulations containing cationic lipids with shorter alkyl tails. LYRs with siRNA against endogenous luciferase and GAPDH were successful in silencing these genes in 3 cell lines (1HAEo- human airway epithelial, B104 rat neuroblastoma, Neuro2A-Luc mouse neuroblastoma) in vitro with 80% efficiency, similar in efficiency to Lipofectamine 2000. Confocal microscopy analysis with LYRs containing fluorescently labelled siRNA (Cy3) showed that the siRNA was located in the perinuclear region of the cytoplasm, where the RNA-induced silencing complex (RISC) is likely to be found. The LYR formulations may have applications for the further development of siRNA-based therapeutics.

Original languageEnglish
Pages (from-to)6302-15
Number of pages14
JournalBiomaterials
Volume32
Issue number26
DOIs
Publication statusPublished - 30 May 2011

Fingerprint

Liposomes
RNA
Peptides
Small Interfering RNA
Neuroblastoma
Transfection
Tail
Lipids
RNA-Induced Silencing Complex
Genes
Cells
Gene Knockdown Techniques
Cell Line
Confocal microscopy
Gene Silencing
RNA Interference
Luciferases
Confocal Microscopy
Nanoparticles
Heparin

Keywords

  • Animals
  • Cell Line
  • Cell Line, Tumor
  • Cell Proliferation
  • Gene Silencing/physiology
  • Heparin/chemistry
  • Humans
  • Liposomes/chemistry
  • Microscopy, Confocal
  • Microscopy, Electron
  • Microscopy, Electron, Transmission
  • Nanoconjugates/chemistry
  • Peptides/chemistry
  • RNA Interference
  • RNA, Small Interfering/administration & dosage
  • Rats

Cite this

Tagalakis, Aristides D ; He, Lin ; Saraiva, Luisa ; Gustafsson, Kenth T ; Hart, Stephen L. / Receptor-targeted liposome-peptide nanocomplexes for siRNA delivery. In: Biomaterials. 2011 ; Vol. 32, No. 26. pp. 6302-15.
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Receptor-targeted liposome-peptide nanocomplexes for siRNA delivery. / Tagalakis, Aristides D; He, Lin; Saraiva, Luisa; Gustafsson, Kenth T; Hart, Stephen L.

In: Biomaterials, Vol. 32, No. 26, 30.05.2011, p. 6302-15.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Receptor-targeted liposome-peptide nanocomplexes for siRNA delivery

AU - Tagalakis, Aristides D

AU - He, Lin

AU - Saraiva, Luisa

AU - Gustafsson, Kenth T

AU - Hart, Stephen L

PY - 2011/5/30

Y1 - 2011/5/30

N2 - RNA interference induced by double-stranded, small interfering RNA (siRNA) molecules has attracted great attention as a genetic therapeutic approach. Despite major advances in this field, new nanoparticle formulations are required for in vivo delivery of siRNA, particularly for tissue-specific delivery of siRNA reagents. We have developed and optimized LYR nanocomplex formulations for siRNA delivery that consist of a liposome (DOTMA/DOPE; L) and a targeting peptide (K₁₆GACYGLPHKFCG; Y) which self-assemble on mixing at optimal ratios with siRNA (R). Biophysical measurements indicated that LYR nanocomplexes were strongly cationic, mainly spherical particles of less than 100 nm. These formulations packaged and protected siRNA on incubation with RNAseA with >90% intact siRNA recovery. In addition, intact siRNA was recovered from LYRs upon heparin treatment. A critical synergy was observed between the lipid and peptide components for LYR particle stability and transfection efficiency. To evaluate targeting, transfections were compared with non-targeted formulations containing K₁₆ with no targeting ligand. Gene knockdown efficiencies with targeted formulations were more than two-fold better in all cell lines tested (p < 0.01). LYR formulations with liposomes containing DOTMA, which has an 18-carbon (C18) alkyl tail, were significantly better in silencing than formulations containing cationic lipids with shorter alkyl tails. LYRs with siRNA against endogenous luciferase and GAPDH were successful in silencing these genes in 3 cell lines (1HAEo- human airway epithelial, B104 rat neuroblastoma, Neuro2A-Luc mouse neuroblastoma) in vitro with 80% efficiency, similar in efficiency to Lipofectamine 2000. Confocal microscopy analysis with LYRs containing fluorescently labelled siRNA (Cy3) showed that the siRNA was located in the perinuclear region of the cytoplasm, where the RNA-induced silencing complex (RISC) is likely to be found. The LYR formulations may have applications for the further development of siRNA-based therapeutics.

AB - RNA interference induced by double-stranded, small interfering RNA (siRNA) molecules has attracted great attention as a genetic therapeutic approach. Despite major advances in this field, new nanoparticle formulations are required for in vivo delivery of siRNA, particularly for tissue-specific delivery of siRNA reagents. We have developed and optimized LYR nanocomplex formulations for siRNA delivery that consist of a liposome (DOTMA/DOPE; L) and a targeting peptide (K₁₆GACYGLPHKFCG; Y) which self-assemble on mixing at optimal ratios with siRNA (R). Biophysical measurements indicated that LYR nanocomplexes were strongly cationic, mainly spherical particles of less than 100 nm. These formulations packaged and protected siRNA on incubation with RNAseA with >90% intact siRNA recovery. In addition, intact siRNA was recovered from LYRs upon heparin treatment. A critical synergy was observed between the lipid and peptide components for LYR particle stability and transfection efficiency. To evaluate targeting, transfections were compared with non-targeted formulations containing K₁₆ with no targeting ligand. Gene knockdown efficiencies with targeted formulations were more than two-fold better in all cell lines tested (p < 0.01). LYR formulations with liposomes containing DOTMA, which has an 18-carbon (C18) alkyl tail, were significantly better in silencing than formulations containing cationic lipids with shorter alkyl tails. LYRs with siRNA against endogenous luciferase and GAPDH were successful in silencing these genes in 3 cell lines (1HAEo- human airway epithelial, B104 rat neuroblastoma, Neuro2A-Luc mouse neuroblastoma) in vitro with 80% efficiency, similar in efficiency to Lipofectamine 2000. Confocal microscopy analysis with LYRs containing fluorescently labelled siRNA (Cy3) showed that the siRNA was located in the perinuclear region of the cytoplasm, where the RNA-induced silencing complex (RISC) is likely to be found. The LYR formulations may have applications for the further development of siRNA-based therapeutics.

KW - Animals

KW - Cell Line

KW - Cell Line, Tumor

KW - Cell Proliferation

KW - Gene Silencing/physiology

KW - Heparin/chemistry

KW - Humans

KW - Liposomes/chemistry

KW - Microscopy, Confocal

KW - Microscopy, Electron

KW - Microscopy, Electron, Transmission

KW - Nanoconjugates/chemistry

KW - Peptides/chemistry

KW - RNA Interference

KW - RNA, Small Interfering/administration & dosage

KW - Rats

U2 - 10.1016/j.biomaterials.2011.05.022

DO - 10.1016/j.biomaterials.2011.05.022

M3 - Article

C2 - 21624650

VL - 32

SP - 6302

EP - 6315

JO - Biomaterials

JF - Biomaterials

SN - 0142-9612

IS - 26

ER -